Anti-tubercular drugs: compositions and methods

ABSTRACT

Methods and compositions for treating disease caused by infectious agents, particularly tuberculosis. In particular, methods and compositions comprising substituted ethylene diamines for the treatment of infectious diseases are provided. In one embodiment, these methods and compositions are used for the treatment of mycobacterial infections, including, but not limited to, tuberculosis. In certain embodiments, the present invention comprises compositions comprising novel substituted ethylene diamine compounds further comprising antitubercular agents such as rifampicin, isoniazid, pyrazinamide and ethambutol.

FIELD OF INVENTION

The present invention relates to methods and compositions for treatinginfectious disease and disease caused by microorganisms, particularlytuberculosis. The present invention also relates to methods andcompositions having improved anti-mycobacterial activity, namelycompositions comprising novel substituted ethylene diamine compounds. Incertain embodiments, the present invention comprises compositionscomprising novel substituted ethylene diamine compounds furthercomprising antitubercular agents such as rifampicin, isoniazid,pyrazinamide, moxifloxacin and ethambutol.

BACKGROUND OF THE INVENTION

Mycobacterial infections often manifest as diseases such astuberculosis. Human infections caused by mycobacteria have beenwidespread since ancient times, and tuberculosis remains a leading causeof death today. Although the incidence of the disease declined, inparallel with advancing standards of living, since the mid-nineteenthcentury, mycobacterial diseases still constitute a leading cause ofmorbidity and mortality in countries with limited medical resources.Additionally, mycobacterial diseases can cause overwhelming,disseminated disease in immunocompromised patients. In spite of theefforts of numerous health organizations worldwide, the eradication ofmycobacterial diseases has never been achieved, nor is eradicationimminent. Nearly one third of the world's population is infected withmycobacterium tuberculosis complex, commonly referred to as tuberculosis(TB), with approximately 8 million new cases, and two to three milliondeaths attributable to TB yearly. Tuberculosis (TB) is the cause of thelargest number of human deaths attributable to a single etiologic agent(see Dye et al., J. Am. Med. Association, 282, 677-686, (1999); and 2000WHO/OMS Press Release).

After decades of decline, TB is now on the rise. In the United States,up to 10 million individuals are believed to be infected. Almost 28,000new cases were reported in 1990, constituting a 9.4 percent increaseover 1989. A sixteen percent increase in TB cases was observed from 1985to 1990. Overcrowded living conditions and shared air spaces areespecially conducive to the spread of TB, contributing to the increasein instances that have been observed among prison inmates, and among thehomeless in larger U.S. cities. Approximately half of all patients with“Acquired Immune Deficiency Syndrome” (AIDS) will acquire amycobacterial infection, with TB being an especially devastatingcomplication. AIDS patients are at higher risks of developing clinicalTB, and anti-TB treatment seems to be less effective than in non-AIDSpatients. Consequently, the infection often progresses to a fataldisseminated disease.

Mycobacteria other than M. tuberculosis are increasingly found inopportunistic infections that plague the AIDS patient. Organisms fromthe M. avium-intracellulare complex (MAC), especially serotypes four andeight, account for 68% of the mycobacterial isolates from AIDS patients.Enormous numbers of MAC are found (up to 10¹⁰ acid-fast bacilli per gramof tissue), and consequently, the prognosis for the infected AIDSpatient is poor.

The World Health Organization (WHO) continues to encourage the battleagainst TB, recommending prevention initiatives such as the “ExpandedProgram on Immunization” (EPI), and therapeutic compliance initiativessuch as “Directly Observed Treatment Short-Course” (DOTS). For theeradication of TB, diagnosis, treatment, and prevention are equallyimportant. Rapid detection of active TB patients will lead to earlytreatment by which about 90% cure is expected. Therefore, earlydiagnosis is critical for the battle against TB. In addition,therapeutic compliance will ensure not only elimination of infection,but also reduction in the emergence of drug-resistance strains.

The emergence of drug-resistant M. tuberculosis is an extremelydisturbing phenomenon. The rate of new TB cases proven resistant to atleast one standard drug increased from 10 percent in the early 1980's to23 percent in 1991. Compliance with therapeutic regimens, therefore, isalso a crucial component in efforts to eliminate TB and prevent theemergence of drug resistant strains. Equally important is thedevelopment of new therapeutic agents that are effective as vaccines,and as treatments, for disease caused by drug resistant strains ofmycobacteria.

Multidrug-resistant tuberculosis (MDR TB) is a form of tuberculosis thatis resistant to two or more of the primary drugs used for the treatmentof tuberculosis. Resistance to one or several forms of treatment occurswhen bacteria develop the ability to withstand antibiotic attack andrelay that ability to their progeny. Since an entire strain of bacteriainherit this capacity to resist the effects of various treatments,resistance can spread from one person to another.

The World Health Organization estimates that up to 50 million personsworldwide may be infected with drug resistant strains of tuberculosis.Also, 300,000 new cases of MDR-TB are diagnosed around the world eachyear and 79 percent of the MDR-TB cases now show resistance to three ormore drugs routinely used to treat tuberculosis.

In 2003, the Centre for Disease control (CDC) reported that 7.7 percentof tuberculosis cases in the U.S. were resistant to isoniazid, a firstline drug used to treat Tuberculosis. The CDC also reported that 1.3percent of tuberculosis cases in the U.S. were resistant to bothisoniazid and rifampin. Rifampin is the drug most commonly used withisoniazid.

Clearly, the possibility of drug resistant strains of tuberculosis thatdevelop during or before treatment are a major concern to healthorganizations and heath care practitioners. Drugs used in the treatmentof tuberculosis include, but are not limited to, Ethambutol,Pyrazinamide, Streptomycin, Isoniazid, Moxifloxacin and Rifampin. Theexact course and duration of treatment can be tailored to a specificindividual, however several strategies are well known to those skilledin the art.

Although over 37 species of mycobacteria have been identified, more than95% of all human infections are caused by six species of mycobacteria:M. tuberculosis, M. avium intracellulare, M. kansasii, M. fortuitum, M.chelonae, and M. leprae. The most prevalent mycobacterial disease inhumans is tuberculosis (TB) which is predominantly caused bymycobacterial species comprising M. tuberculosis, M. bovis, or M.africanum (Merck Manual 1992). Infection is typically initiated by theinhalation of infectious particles which are able to reach the terminalpathways in lungs. Following engulfment by alveolar macrophages, thebacilli are able to replicate freely, with eventual destruction of thephagocytic cells. A cascade effect ensues wherein destruction of thephagocytic cells causes additional macrophages and lymphocytes tomigrate to the site of infection, where they too are ultimatelyeliminated. The disease is further disseminated during the initialstages by the infected macrophages which travel to local lymph nodes, aswell as into the blood stream and other tissues such as the bone marrow,spleen, kidneys, bone and central nervous system. (See Murray et al.Medical Microbiology, The C.V. Mosby Company 219-230 (1990)).

There is still no clear understanding of the factors which contribute tothe virulence of mycobacteria. Many investigators have implicated lipidsof the cell wall and bacterial surface as contributors to colonymorphology and virulence. Evidence suggests that C-mycosides, on thesurface of certain mycobacterial cells, are important in facilitatingsurvival of the organism within macrophages. Trehalose 6,6′ dimycolate,a cord factor, has been implicated for other mycobacteria.

The interrelationship of colony morphology and virulence is particularlypronounced in M. avium. M. avium bacilli occur in several distinctcolony forms. Bacilli which grow as transparent, or rough, colonies onconventional laboratory media are multiplicable within macrophages intissue culture, are virulent when injected into susceptible mice, andare resistant to antibiotics. Rough or transparent bacilli, which aremaintained on laboratory culture media, often spontaneously assume anopaque R colony morphology, at which time they are not multiplicable inmacrophages, are avirulent in mice, and are highly susceptible toantibiotics. The differences in colony morphology between thetransparent, rough and opaque strains of M. avium are almost certainlydue to the presence of a glycolipid coating on the surface oftransparent and rough organisms which acts as a protective capsule. Thiscapsule, or coating, is composed primarily of C-mycosides whichapparently shield the virulent M. avium organisms from lysosomal enzymesand antibiotics. By contrast, the non-virulent opaque forms of M. aviumhave very little C-mycoside on their surface. Both the resistance toantibiotics and the resistance to killing by macrophages have beenattributed to the glycolipid barrier on the surface of M. avium.

Diagnosis of mycobacterial infection is confirmed by the isolation andidentification of the pathogen, although conventional diagnosis is basedon sputum smears, chest X-ray examination (CXR), and clinical symptoms.Isolation of mycobacteria on a medium takes as long as four to eightweeks. Species identification takes a further two weeks. There areseveral other techniques for detecting mycobacteria such as thepolymerase chain reaction (PCR), mycobacterium tuberculosis direct test,or amplified mycobacterium tuberculosis direct test (MTD), and detectionassays that utilize radioactive labels.

One diagnostic test that is widely used for detecting infections causedby M. tuberculosis is the tuberculin skin test. Although numerousversions of the skin test are available, typically one of twopreparations of tuberculin antigens are used: old tuberculin (OT), orpurified protein derivative (PPD). The antigen preparation is eitherinjected into the skin intradermally, or is topically applied and isthen invasively transported into the skin with the use of a multipronginoculator (Tine test). Several problems exist with the skin testdiagnosis method. For example, the Tine test is not generallyrecommended because the amount of antigen injected into the intradermallayer cannot be accurately controlled. (See Murray et al. MedicalMicrobiology, The C.V. Mosby Company 219-230 (1990)).

Although the tuberculin skin tests are widely used, they typicallyrequire two to three days to generate results, and many times, theresults are inaccurate since false positives are sometimes seen insubjects who have been exposed to mycobacteria, but are healthy. Inaddition, instances of mis-diagnosis are frequent since a positiveresult is observed not only in active TB patients, but also in personsvaccinated with Bacille Calmette-Guerin (BCG), and those who had beeninfected with mycobacteria, but have not developed the disease. It ishard therefore, to distinguish active TB patients from the others, suchas household TB contacts, by the tuberculin skin test. Additionally, thetuberculin test often produces a cross-reaction in those individuals whowere infected with mycobacteria other than M. tuberculosis (MOTT).Therefore, diagnosis using the skin tests currently available isfrequently subject to error and inaccuracies.

The standard treatment for tuberculosis caused by drug-sensitiveorganisms is a six-month regimen consisting of four drugs given for twomonths, followed by two drugs given for four months. The two mostimportant drugs, given throughout the six-month course of therapy, areisoniazid and rifampin. Although the regimen is relatively simple, itsadministration is quite complicated. Daily ingestion of eight or ninepills is often required during the first phase of therapy; a dauntingand confusing prospect. Even severely ill patients are often symptomfree within a few weeks, and nearly all appear to be cured within a fewmonths. If the treatment is not continued to completion, however, thepatient may experience a relapse, and the relapse rate for patients whodo not continue treatment to completion is high. A variety of forms ofpatient-centered care are used to promote adherence with therapy. Themost effective way of ensuring that patients are taking their medicationis to use directly observed therapy, which involves having a member ofthe health care team observe the patient take each dose of each drug.Directly observed therapy can be provided in the clinic, the patient'sresidence, or any mutually agreed upon site. Nearly all patients whohave tuberculosis caused by drug-sensitive organisms, and who completetherapy will be cured, and the risk of relapse is very low (“EndingNeglect: The Elimination of Tuberculosis in the United States” ed. L.Geiter Committee on the Elimination of Tuberculosis in the United StatesDivision of Health Promotion and Disease Prevention, Institute ofMedicine. Unpublished.)

Although the FDA approved a medication that combines the three maindrugs (isoniazid, rifampin, and pyrazinamide) used to treat tuberculosisinto one pill. Thereby reducing the number of pills a patient has totake each day and making it impossible for the patient to take only oneof the three medications, a common path to the development of MDR-TB,there is still a need in the art to treat tuberculosis, especially inthose cases wherein the tuberculosis strain is drug resistant.

What is needed are effective therapeutic regimens that include improvedvaccination and treatment protocols. Currently available therapeuticsare no longer consistently effective as a result of the problems withtreatment compliance, and these compliance problems contribute to thedevelopment of drug resistant mycobacterial strains.

Furthermore, there is a need in the art for novel compositions andmethods that are effective against infectious disease. Moreparticularly, there is a need for novel compositions and methods for theeffective treatment of Mycobacterial disease.

Ethambutol (EMB) is a widely used antibiotic for the treatment of TB,with over 300 million doses delivered for tuberculosis therapy in 1988.

Ethambutol, developed by Lederle Laboratories in the 1950s, has lowtoxicity and is a good pharmacokinetic. However, ethambutol has arelatively high Minimum Inhibition Concentration (MIC) of about 5 μg/ml,and can cause optic neuritis. Thus, there is an increasing need for new,and more effective, therapeutic compositions (See for example, U.S. Pat.No. 3,176,040, U.S. Pat. No. 4,262,122; U.S. Pat. No. 4,006,234; U.S.Pat. No. 3,931,157; U.S. Pat. No. 3,931,152; U.S. Re. 29,358; andHäusler et al., Bioorganic & Medicinal Chemistry Letters 11 (2001)1679-1681). In the decoder years since the discovery of the beneficialeffects of ethambutol, few pharmacological advances in TB treatment havebeen developed. Moreover, with the combined emergence of drug resistantstrains, and the more prevalent spread of mycobacterial disease, it isbecoming seriously apparent that new therapeutic compositions arecrucial in the fight against tuberculosis.

Clearly effective therapeutic regimens that include improved vaccinationand treatment protocols are needed. A therapeutic vaccine that wouldprevent the onset of tuberculosis, and therefore eliminate the need fortherapy is desirable. Although currently available therapeutics such asethambutol are effective, the emergence of drug resistant strains hasnecessitated new formulations and compositions that are more versatilethan ethambutol. Currently available therapeutics are no longerconsistently effective as a result of the problems with treatmentcompliance, lending to the development of drug resistant mycobacterialstrains. What is needed are new anti-tubercular drugs that providehighly effective treatment, and shorten or simplify tuberculosischemotherapy.

SUMMARY OF THE INVENTION

The present invention comprises methods and compositions comprisingethylene diamine compounds effective for the treatment of infectiousdisease. The present invention also provides methods and compositionscomprising substituted ethylene diamines having improvedanti-mycobacterial activity, including substituted ethylene diamineshaving improved anti-tuberculosis activity.

The present invention contemplates substituted ethylene diamines, whichcan derive from a variety of amine compounds. In the present invention,the substituted ethylene diamines are based on the following structure.

The substituted ethylene diamine compounds described herein aresynthesized and screened for activity as follows. A chemical library ofsubstituted ethylene diamines is prepared on a solid polystyrene supportusing split and pool technologies. This technique allows for thesynthesis of a diverse set of substituted ethylene diamines. Thesediamines are screened for anti-TB activity using in vitro, biologicalassays, including a High-Throughput Screening (HTS) assay, based on therecently completed genomic sequence of M. tuberculosis, and a MinimumInhibition Concentration (MIC) assay.

The methods and compositions described herein comprise substitutedethylene diamines that are effective against disease caused byinfectious organisms, including, but not limited to, bacteria andviruses. One embodiment of the invention provides methods andcompositions comprising substituted ethylene diamines that are effectiveagainst mycobacterial disease. Another embodiment of the inventionprovides methods and compositions comprising substituted ethylenediamines that have MIC of 50 μM or lower for mycobacterial disease.Another embodiment of the present invention comprises substitutedethylene diamines that have an MIC of 25 μM or lower for mycobacterialdisease. Yet another embodiment of the present invention comprisessubstituted ethylene diamines that have an MIC of 12.5 μM or lower formycobacterial disease. Another embodiment of the present inventioncomprises substituted ethylene diamines that have an MIC of 5 μM orlower for mycobacterial disease In another embodiment of the presentinvention, the methods and compositions comprise substituted ethylenediamines with HTS Luc activity of 10% or greater. In yet anotherembodiment of the present invention, the methods and compositionscomprise substituted ethylene diamines, wherein one amine group isderived from a primary amine, and wherein the other amine group isderived from a primary or secondary amine. In another embodiment of thepresent invention, the methods and compositions comprise substitutedethylene diamines, wherein one amine is derived fromcis-(−)myrtanylamine, cyclooctylamine, 2,2-diphenylethylamine,3,3-diphenylpropylamine, (+)-bornylamine, 1-adamantanemethylamine,(+)-isopinocampheylamine; or isopinocampheylaminc.

The present invention contemplates various salt complexes and othersubstituted derivatives of the substituted ethylene diamines. Thepresent invention also contemplates enantiomers and other stereoisomersof the substituted ethylene diamines and their substituted derivatives.The present invention further contemplates treatment for animals,including, but not limited to, humans.

In addition the present invention contemplates compositions comprisingnovel substituted ethylene diamine compounds further comprisingantitubercular agents including, but not limited to, rifampicin,isoniazid, pyrazinamide, moxifloxacin and ethambutol and analoguesthereof.

Accordingly, it is an object of the present invention to provide methodsand compositions for the treatment and prevention of diseases caused bymicroorganisms

Accordingly, it is an object of the present invention to provide methodsand compositions for the treatment and prevention of infectiousdiseases.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of mycobacterial disease,including but not limited to, tuberculosis.

Yet another object of the present invention is to provide methods andcompositions for the treatment and prevention of infectious diseasesusing compositions comprising substituted ethylene diamines.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of mycobacterial diseaseusing compositions comprising substituted ethylene diamines.

Still another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising substituted ethylene diamines.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising substituted ethylene diamines, wherein thediamine has an MIC of 50 μM, or less.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising substituted ethylene diamines, wherein thediamine has an MIC of 25 μM, or less.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising substituted ethylene diamines, wherein thediamine has an MIC of 12.5 μM, or less.

Yet another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising substituted ethylene diamines, wherein thediamine has an MIC of 5 μM, or less.

Yet another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising substituted ethylene diamines, wherein thediamine has HTS/Luc activity of 10% or greater.

Another object of the present invention is to provide methods andcompositions for the treatment and prevention of tuberculosis usingcompositions comprising substituted ethylene diamines, wherein one aminegroup is derived from a primary amine, and the other amine group isderived from a primary or secondary amine.

Yet another object of the present invention is to provide methods andcompositions for the treatment and/or prevention of tuberculosis usingcompositions comprising substituted ethylene diamines, wherein one amineis derived from cis-(−)myrtanylamine, cyclooctylamine,2,2-diphenylethylamine, 3,3-diphenylpropylamine, (+)-bornylamine,1-adamantanemethylamine, (+)-isopinocampheylamine; or(−)-isopinocampheylamine.

Yet another object of the present invention is to provide compositionfor the therapeutic formulation for the treatment and prevention ofmycobacterial disease.

Another object of the present invention is to provide compositions fortherapeutic formulations for the treatment and prevention ofmycobacterial disease caused by mycobacterial species comprising M.tuberculosis complex, M. avium intracellulare, M. kansarii, M.fortuitum, M. chelonoe, M. leprae, M. africanum, M. microti, M. bovisBCG or M. bovis.

Still another object of the present invention is to provide compositionsand methods for the treatment or prevention of infectious disease causedby Mycobacterium-fortuitum, Mycobacterium marinum, Helicobacter pylori,Streptococcus pneumoniae and Candida albicans.

Another object of the instant invention is to provide one or more novelcompounds in a combination therapy to provide a synergistic effect thatis active against mycobacterial disease.

A further object of the instant invention is to provide compositionscomprising novel substituted ethylene diamine compounds furthercomprising antitubercular agents including, but not limited to,rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol andanalogues thereof.

Another object of the present invention is to provide combinationtherapy for infectious disease comprising substituted ethylene diaminesand antitubercular agents.

An additional object of the present invention is to provide combinationtherapy for mycobacterial disease comprising substituted ethylenediamines and antitubercular agents including, but not limited to,rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol andanalogues thereof either singularly or in combination.

Yet another object of the present invention is to provide combinationtherapy for mycobacterial disease such as tuberculosis comprisingsubstituted ethylene diamines and antitubercular agents including, butnot limited to, rifampicin, isoniazid, pyrazinamide, moxifloxacin andethambutol and analogues thereof either singularly or in combination.

Another object of the present invention is to provide combinationtherapy for mycobacterial disease such as tuberculosis comprisingsubstituted ethylene diamines such as SQ 109, SQ 73 or SQ 609 andantitubercular agents including, but not limited to, rifampicin,isoniazid, pyrazinamide, moxifloxacin and ethambutol and analoguesthereof either singularly or in combination.

Another object of the present invention is to provide combinationtherapy for mycobacterial disease such as tuberculosis comprisingsubstituted ethylene diamines such as SQ 109, SQ 73 or SQ 609 andantitubercular agents including, but not limited to, rifampicin, andrifampicin analogues such as rifapentine, rifalazil and rifabutin.

Yet another object of the instant invention is to provide one or morenovel compounds in combination with a drug to provide a synergisticeffect active against mycobacterial disease.

Another object of the instant invention is to provide one or more novelcompounds in combination with a standard tuberculosis drug to provide asynergistic effect active against mycobacterial disease.

It is a further objective that the instant invention provide novelmethods of treatment wherein one or more novel compounds are used incombination with at least one known drug to provide a synergisticeffect, by which to treat or prevent infectious disease.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents a flow chart schematic showing various solid supportsyntheses used to prepare substituted ethylene diamines.

FIGS. 2( a)-2(ac) provide chemical structures of a variety of primaryamines.

FIGS. 3( a)-3(f) provide chemical structures of a variety of acyclicsecondary amines.

FIGS. 4( a)-4(i) provide chemical structures of a variety of cyclicsecondary amines.

FIG. 5 represents a flow schematic for a representative reaction pool often substituted ethylene diamines.

FIG. 6 is a graph of Luminescence Count per Second (LCPS) versusconcentration showing HTS Luc assay results for pooled substitutedethylene diamine compounds.

FIG. 7 is a graph of LCPS versus concentration showing HTS Luc assayresults for individual substituted ethylene diamine compounds.

FIG. 8 is a graph of LCPS versus concentration showing HTS Luc assayresults for individual substituted ethylene diamine compounds.

FIG. 9 is a bar graph providing a summary of MIC activities for discretesubstituted ethylene diamines.

FIG. 10 is a bar graph providing a summary of Luciferase activity ofdiscrete substituted ethylene diamines with at least 10% activity inreference to ethambutol at 3.1 μM.

FIG. 11 is a bar graph showing the frequency of occurrences of theselected amine monomers in the substituted ethylene diamine compoundsthat were active against TB. Amine monomers are represented by theirnumerical designations.

FIG. 12 represents a flow schematic showing a synthesis ofN-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine (compound 109).

FIG. 13 is a flow schematic showing a synthesis ofN-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine ashydrochloride (compound 59).

FIG. 14 is a mass spec profile for one representative sample wellcontaining pooled substituted ethylene diamine compounds.

FIG. 15 is a mass spec profile for compound 109,N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine.

FIG. 16 is a proton NMR profile for compound 109,N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine.

FIG. 17 is a bar graph of data from a Colony Forming Units/Lung(CFU/Lung) study showing CFU/Lung growth over time in days for variouscompounds.

FIG. 18 is a bar graph of data from a CFU/Lung study showing CFU/Lunggrowth over time in days for various compounds.

FIG. 19 is a bar graph of data from a CFU/Lung study showing CFU/Lunggrowth over time in days for various compounds.

FIG. 20 is a bar graph of data from a lesion study showing visiblelesions over time after treatment with various compounds.

FIG. 21 provides a schematic demonstrating the identification of a drugcandidate.

FIG. 22 provides the compounds tested for in vivo efficacy.

FIG. 23 is a graph showing the results of in vivo studies of compounds73 and 109 at 1 and 10 mg/kg doses (spleen).

FIG. 24 is a graph showing the results of in vivo studies of compounds73 and 109 at 1 and 10 mg/kg doses (lungs).

FIG. 25 is a graph showing in vivo studies of compounds 59 and 111 at 1and 10 mg/kg doses (spleen).

FIG. 26 is a graph showing in vivo studies of compounds 59 and 111 at 1and 10 mg/kg doses (lungs).

FIG. 27 is a graph showing the results of efficacy testing of thecompounds 58, 73, 109, and 111 in C57BL.6 mice infected with M.tuberculosis H37Rv (spleen). Mice were infected i.v. with 5×10⁶ CFU M.tuberculosis H37Rv; treatment with drugs started 18 days followinginfection. EC-EC—early control, CFU in lungs of mice at the day ofchemotherapy start. Mice received: 1—untreated mice, 2—INH (25 mg/kg),3—EMB (100 mg/kg), 4—comp. 109 (25 mg/kg), 4*—comp.109 (10 mg/kg),4**—comp. 109 (0.1 mg/kg), 5—comp. 58 (25 mg/kg), 6—comp.73 (25 mg/kg),7—comp. 111 (25 mg/kg).

FIG. 28 is a graph showing the results of efficacy testing of thecompounds 58, 73, 109, and 111 in C57BL.6 mice infected with M.tuberculosis H37Rv (lungs). Mice were infected i.v. with 5×10⁶ CFU M.tuberculosis H37Rv; treatment with drugs started 18 days followinginfection. EC-EC—early control, CFU in lungs of mice at the day ofchemotherapy start. Mice received: 1—untreated mice, 2—INH (25 mg/kg),3—EMB (100 mg/kg), 4—comp. 109 (25 mg/kg), 4*—comp.109 (10 mg/kg),4**—comp. 109 (0-1 mg/kg), 5—comp. 58 (25 mg/kg), 6—comp.73 (25 mg/kg),7—comp. 111 (25 mg/kg).

FIG. 29 provides LC/MS data of tested compounds.

FIG. 30 provides a graph showing results of PK studies with a cassettedosing of tested compounds to mice. Oral delivery. Compound NSC 722039in the study reads as the compound 37, NSC 722040—compound 59, NSC722041—compound 109.

FIG. 31 provides a graph showing results of PK studies with a cassettedosing of tested compounds to mice. Peritoneal delivery. Compound NSC722039 in the study reads as the compound 37, NSC 722040—compound 59,NSC 722041—compound 109.

FIG. 32 provides a graph showing results of PK studies with a cassettedosing of tested compounds to mice. Intravenous delivery. Compound NSC722039 in the study reads as the compound 37, NSC 722040—compound 59,NSC 722041—compound 109.

FIG. 33 provides a graph showing the results of PK Studies of thecompound 109 in mice.

FIG. 34. Tissue distribution of 109 in mice (i.v., 3 mg/kg).

FIG. 35. Tissue distribution of 109 in mice (p.o., 25 mg/kg).

FIG. 36 Metabolism of the compound 109 in mouse urine.

FIG. 37. No glucoronidation metabolites of 109 were found in mouseurine.

FIG. 38. Binding assays for compound 109.

FIG. 39. Binding assays for reference compound.

FIG. 40. Data summary for compound 109

FIG. 41. Scheme 1. Synthesis of 100,000 compound library of ethambutolanalogues on solid support.

FIG. 41. Scheme 2. Attempts to synthesize SQBisAd on solid support.

FIG. 42 provides structures of representative targeted diamines preparedvia acylation by amino acids.

FIG. 43 provides Table 25 summarizing data for synthesized plates ofdiamines for the prepared library of targeted 20,000 ethambutol analogs.

FIG. 44 provides Scheme 5 showing the synthesis of the diamine libraryusing amino acids as linkers.

FIG. 45 provides a schematic showing the occurrence of amine monomers inthe hits that were generated in the original 100,000 compound library ofEMB analogs.

FIG. 46 provides a schematic showing structural diversity among primaryamines.

FIG. 47 provides Table 26 listing the amino acids that were used in thepreparation of the diamine library.

FIG. 48 provides carbonyl compounds used as reagents in the synthesis ofthe diamine library.

FIG. 49 provides Table 27 showing carbonyl compounds used in themasterplate for the synthesis of the diamine library.

FIG. 50 provides representative examples of MIC and Lux data for thediamine library.

FIG. 51 provides a schematic showing the occurrence of alkylatingmonomers in final diamine products with anti-TB activity.

FIG. 52 provides the layout of a representative 96-well deconvolutionplate.

FIG. 53 provides a list of compound hits and structures for the modifiedlinker diamine library.

FIG. 54 provides a graph showing the results of in vivo activity ofRifampin, compound 109 (SQ109) or Isoniazid (INH). The study was carriedout using BACTEC system. MIC for RIF was 0.2 ug/ml, SQ109 (compound 109)0.32 ug/ml, and INH 0.025 ug/ml.

FIG. 55 is a graph showing the results of in vivo studies in mice usinga rapid model, wherein body weight of mice is used as a marker toestimate drug efficacy. The results are of a 21 day combination therapystudy in the rapid model. C3H female mice were infected i.v. with 10⁶CFU M. tuberculosis H37Rv (Pasteur). 7 days following inoculationchemotherapy was initiated and continued for 2 weeks (5 days/week). Micetreated with a single drug, uninfected, and infected untreated placebowere used as controls. Rif at 2 mg/kg, INH at 1 mg/kg, EMB at 10 mg/kg,Moxi at 10 mg/kg, PZA at 50 mg/kg. Body weight of mice monitored fromday 0 through day 21.

FIGS. 56A and 56B are graphs disclosing dynamics in body weight (FIG.56A) and the mortality data (FIG. 56B) of H37Rv infected animals treatedwith SQ109 (10 mg/kg), Rifampin (2 mg/kg), and SQ 109 (compound 109) (10mg/kg)-Rif(2 mg/kg) combination, and of the placebo (infectednon-treated) in the rapid model. C3H female mice were infected i.v. with10⁶ CFU M. tuberculosis H37Rv (Pasteur). 7 days following inoculationchemotherapy was initiated and continued for 2 weeks (5 days/week). Bodyweight of mice monitored from day 0 through the end of chemotherapy (day21).

FIG. 57 is a graph showing the dynamics of body weight of mice treatedwith combination, Rif-EMB. Dynamics in body weight of H37Rv infectedanimals treated with Ethambutol (10 mg/kg), Rifampin (2 mg/kg), andEthambutol (10 mg/kg)-Rif (2 mg/kg) combination, and of the placebo. C3Hfemale mice were infected i.v. with 10⁶ CFU M. tuberculosis H37Rv(Pasteur). 7 days following inoculation chemotherapy was initiated andcontinued for 2 weeks (5 days/week). Body weight of mice were monitoredfrom day 0 through the end of chemotherapy (day 21).

FIG. 58 provides a graph showing the in vivo efficacy studies ofcompound 109 in combination with Rifampin in a mouse model of chronictuberculosis infection. C57BL/6 female mice were inoculated i.v. with10⁵ CFU M. tuberculosis H37Rv. Chemotherapy was initiated three weeksfollowing the infection and continued for 4 weeks. At the end oftherapy, mice were sacrificed; lungs homogenates in sterile 2 ml PBSwith 0.05% Tween-80 were plated in 10-fold serial dilutions on 7H10 agardishes, and were incubated at 37° C. CFU were calculated after 3 wk ofgrowth. 16 groups of mice were used (6 mice per group). Compound 109(SQ109) was used at 0, 0.1, 1, and 10 mg/kg; RIF at 0, 1, 2, and 20mg/kg.

FIG. 59 shows the results of compound 109 in a multi-drug intensivephase regime in combination with Rif and INH using the chronic mousemodel. C57BL/6 female mice were inoculated i.v. with 10⁵ CFU M.tuberculosis H37Rv. Chemotherapy was initiated three weeks following theinfection and continued for 5 weeks with time points at 2, 3, 4, and 5weeks. At each timepoint, one group of mice for tested drug combinations(6 mice per group) was sacrificed; lungs homogenates in sterile 2 ml PBSwith 0.05% Tween-80 were plated in 10-fold serial dilutions on 71110agar dishes, and were incubated at 37° C. CFU were calculated after 3 wkof growth. INH was used at 25 mg/kg, RIF at 20 mg/kg, SQ109 (compound109) at 10 mg/kg, EMB at 100 mg/kg. Statistic analysis was done usingthe ANOVA test: significance of any differences was estimated byStudent's T-test and p<0.05 was considered statistically significant. 3weeks * −p=0.001, 4 weeks ** p=0.008, and for 5 weeks *** p=0.005.

FIG. 60 provides results of binding assay as % of Control SpecificBinding for compound 109.

FIG. 61 provides results of binding assay as % inhibition of ControlSpecific Binding for compound 109.

FIG. 62 provide MIC results of Gram-Positive Organisms Tested AgainstSQ-109.

FIGS. 63A and 63B provide MIC results of Gram-Negative Organisms TestedAgainst SQ-109.

FIG. 64 provides MIC results of Anaerobes Tested Against SQ-109.

FIG. 65 provides MIC results of Fungi Tested Against SQ-109.

FIG. 66 provides MIC results of Mycobacteria Tested Against SQ-109.

FIG. 67 provides Table 40 showing synergy quotients for SQ109 tested intwo-drug combinations with INH, STR, EMB, or PZA; and Table 41 showingsynergy quotients for SQ109 tested in combination with RIF.

FIG. 68 provides the growth profile of RIF^(R) M. tuberculosis isolate3185 treated with rifampicin (RIF) in combination with either SQ109 orethambutol (EMB). The Growth Index (GI) of RIF^(R) M. tuberculosisstrain 3185 in BACTEC vials containing various concentrations of RIF incombination with 0.5 MIC SQ109 (a) or 0.5 MIC EMB (b) was monitoreddaily by the BACTEC 460 system. The vials were incubated at 37° C. GIreadings were obtained daily after the first 2 days of the experimentand until the 1:100 inoculum control vial reached a ΔGI value greaterthan 30 at day 8. The MIC for RIF, SQ109, and EMB in Strain 3185 were 24mg/L, 0.32 mg/L, and 2.5 mg/L, respectively.

FIG. 69 provides the growth profile of RIF^(R) M. tuberculosis isolate2482 treated with RIF and SQ 109. The experiment was carried out inBACTEC 460 as described in FIG. 68 legend. GI readings were obtaineddaily until the 1:100 inoculum control vial reached a ΔGI value greaterthan 30 at day 7. The MIC of RIF and SQ109 in Strain 2482 were >100 mg/Land 0.32 mg/L, respectively.

FIG. 70 provides a graph showing the results of a study wherein C57BL/6mice were infected with M. tuberculosis H37Rv and infection was allowedto progress for 21 days. At day 21, mice were treated with INH(25mg/kg)+RIF(20 mg/kg)+PZA(150 mg/kg)+EMB(100 mg/kg) or INH(25mg/kg)+RIF(20 mg/kg)+PZA(1150 mg/kg)+SQ109 (10 mg/kg) for 8 weeks.

FIG. 71 provides a graph showing the synergistic effects of SQ109-Rifcombination. This synergy worked both ways: SQ109 synergisticallyenhanced RIF activity, and RIF synergistically enhanced SQ109 activity.SQ109 at 0.5 MIC showed synergy with RIF at concentrations as low as 0.1MIC. Synergy was also observed when 0.2 MIC SQ109 was combined with 0.5MIC RIF. Interestingly, the combination of both drugs below theireffective concentrations (0.2 MIC SQ109+0.25 MIC RIF) showed an additiveinteraction. In vitro data indicate that SQ109-Rif combination is moreeffective than INH-Rif combination.

FIG. 72 provides a growth profile of RIF^(R) M. tuberculosis isolate2482 treated with RIF and SQ109. The experiment was carried out inBACTEC 460. The MIC of RIF and SQ109 in Strain 2482 were >100 μg/ml and0.32 μg/ml, respectively.

FIG. 73 provides the results of a rapid model, combination therapystudy, day 21. C3H female mice were infected i.v. with 106 CFU M.tuberculosis H37Rv (Pasteur) previously passed through mice. 7 daysfollowing inoculation chemotherapy with anti-TB drugs were initiated andcontinued till day 21.

FIG. 74 provides the structures of SQ109, SQ609, and SQ73.

FIG. 75 provides the results of a study involving chronic TB. C57BL/6female mice were inoculated i.v. with 104 CFU M. tuberculosis H37Rv.Chemotherapy was initiated four weeks following the infection andcontinued for 2 weeks. One group of mice (6 mice per group) was testedfor each control drug and the drug combination. After 2 weeks oftreatment mice were sacrificed; lungs homogenates in sterile 2 ml PBSwith 0.05% Tween-80 were plated in 10-fold serial dilutions on 7H10 agardishes, and were incubated at 37° C. CFU were calculated after 3 wk ofgrowth. INH was used at 25 mg/kg, SQ109 at 10 and 25 mg/kg, SQ609 at 10mg/kg; combination (“Sum” on the chart): SQ109 at 10 mg/kg; SQ609 at 10mg/kg, SQ73 at 5 mg/kg. Statistic analysis was done using the ANOVAtest: significance of any differences was estimated by Student's T-testand p<0.05 was considered statistically significant.

DETAILED DESCRIPTION

The present invention may be understood more readily by reference to thefollowing detailed description of the specific embodiments includedherein. However, although the present invention has been described withreference to specific details of certain embodiments thereof, it is notintended that such details should be regarded as limitations upon thescope of the invention. The entire text of the references mentionedherein are hereby incorporated in their entireties by referenceincluding U.S. patent application Ser. No. 11/145,499, filed Jun. 3,2005, U.S. patent application Ser. No. 10/147,587 filed May 17, 2002,and U.S. Provisional Patent Application Ser. No. 60/381,220 filed May17, 2002.

Mycobacterial infections, such as those causing tuberculosis, oncethought to be declining in occurrence, have rebounded, and againconstitute a serious health threat. Tuberculosis (TB) is the cause ofthe largest number of human deaths attributed to a single etiologicagent with two to three million people infected with tuberculosis dyingeach year. Areas where humans are crowded together, or living insubstandard housing, are increasingly found to have persons affectedwith mycobacteria. Individuals who are immunocompromised are at greatrisk of being infected with mycobacteria and dying from such infection.In addition, the emergence of drug-resistant strains of mycobacteria hasled to treatment problems of such infected persons

Many people who are infected with mycobacteria are poor, or live inareas with inadequate healthcare facilities. As a result of variousobstacles (economical, education levels, etc.), many of theseindividuals are unable to comply with the prescribed therapeuticregimens. Ultimately, persistent non-compliance by these and otherindividuals results in the prevalence of disease. This noncompliance isfrequently compounded by the emergence of drug-resistant strains ofmycobacteria. Effective compositions and vaccines that target variousstrains of mycobacteria are necessary to bring the increasing number oftuberculosis cases under control.

Chemotherapy is a standard treatment for tuberculosis. Some currentchemotherapy treatments require the use of three or four drugs, incombination, administered daily for two months, or administered biweeklyfor four to twelve months. Table 1 lists several treatment schedules forstandard tuberculosis drug regimens.

TABLE 1 Treatment Schedules for Standard TB Drug Regimens. INDUCTIONSTANDARD PHASE CONTINUATION DRUG Dosing PHASE REGIMEN Schedule DURATIONDRUG Dosing Schedule DURATION Isoniazid Daily, DOT 8 weeks Isoniazid2/week, DOT 16 weeks Rifampicin Daily, DOT 8 weeks Rifampicn 2/week, DOT16 weeks Pyrazinamide Daily, DOT 8 weeks Ethambutol or Daily, DOT 8weeks Streptomycin

Decades of misuse of existing antibiotics and poor compliance withprolong and complex therapeutic regimens has led to mutations of themycobacterium tuberculosis and has created an epidemic of drugresistance that threatens tuberculosis control world wide. The vastmajority of currently prescribed drugs, including the front line drugs,such as isoniazid, rifampin, pyrazinamide, ethambutol and streptomycinwere developed from the 1950s to the 1970s. Thus, this earlierdevelopment of tuberculosis chemotherapy did not have at its disposalthe implications of the genome sequence of Mycobacterium tuberculosis,the revolution in pharmaceutical drug discovery of the last decades, andthe use of national drug testing and combinational chemistry.

Consequently, the treatments of drug-resistant M. tuberculosis strains,and latent tuberculosis infections, require new anti-tuberculosis drugsthat provide highly effective treatments, and shortened and simplifiedtuberculosis chemotherapies. Moreover, it is desirable that these drugsbe prepared by a low-cost synthesis, since the demographics of thedisease dictate that cost is a significant factor.

The present invention provides methods and compositions comprising aclass of substituted ethylene diamine compounds effective in treatmentand prevention of disease caused by microorganisms including, but notlimited to, bacteria. In particular, the methods and compositions of thepresent invention are effective in inhibiting the growth of themicroorganism, M. tuberculosis. The methods and compositions of thepresent invention are intended for the treatment of mycobacteriainfections in human, as well as other animals. For example, the presentinvention may be particularly useful for the treatment of cows infectedby M. bovis.

As used herein, the term “tuberculosis” comprises disease states usuallyassociated with infections caused by mycobacteria species comprising M.tuberculosis complex. The term “tuberculosis” is also associated withmycobacterial infections caused by mycobacteria other than M.tuberculosis (MOTT). Other mycobacterial species include M.avium-intracellulare, M. kansarii, M. fortuitum, M. chelonae, M. leprae,M. africanum, and M. microti, M. avium paratuberculosis, M.intracellulare, M. scrofulaceum, M. xenopi, M. marinum, M. ulcerans.

The present invention further comprises methods and compositionseffective for the treatment of infectious disease, including but notlimited to those caused by bacterial, mycological, parasitic, and viralagents. Examples of such infectious agents include the following:staphylococcus, streptococcaceae, neisseriaaceae, cocci,enterobacteriaceae, pseudomonadaceae, vibrionaceae, campylobacter,pasteurellaceae, bordetella, francisella, brucella, legionellaceae,bacteroidaceae, gram-negative bacilli, clostridium, corynebacterium,propionibacterium, gram-positive bacilli, anthrax, actinomyces,nocardia, mycobacterium, Helicobacter pylori, Streptococcus pneumoniae,Candida albicans, treponema, borrelia, leptospira, mycoplasma,ureaplasma, rickettsia, chlamydiae, systemic mycoses, opportunisticmycoses, protozoa, nematodes, trematodes, cestodes, adenoviruses,herpesviruses, poxviruses, papovaviruses, hepatitis viruses,orthomyxoviruses, paramyxoviruses, coronaviruses, picornaviruses,reoviruses, togaviruses, flaviviruses, bunyaviridae, rhabdoviruses,human immunodeficiency virus and retroviruses.

The present invention further provides methods and compositions usefulfor the treatment of infectious disease, including by not limited to,tuberculosis, leprosy, Crohn's Disease, acquired immunodeficiencysyndrome, lyme disease, cat-scratch disease, Rocky Mountain SpottedFever and influenza.

The anti-infective methods and compositions of the present inventioncontain one or more substituted ethylene diamine compounds. Inparticular, these compounds encompass a wide range of substitutedethylene diamine compounds having the following general formula:

where “R₁NH” is typically derived from a primary amine, and “R₂R₃N” istypically derived from a primary or secondary amine. The ethylenediamines of the present invention are prepared by a modular approachusing primary ard secondary amines as building blocks, and coupling theamine moieties with an ethylene linker building block. Representativeprimary amines, acyclic secondary amines, and cyclic secondary aminesare shown in FIGS. 2, 3, and 4, respectively.

Generally, chemical moieties R₁, R₂, and R₃ of the ethylene diaminecompounds of the present invention are independently selected from H,alkyl; aryl; alkenyl; alkynyl; aralkyl; aralkenyl; aralkynyl;cycloalkyl; cycloalkenyl; heteroalkyl; heteroaryl; halide; alkoxy;aryloxy; alkylthio; arylthio; silyl; siloxy; a disulfide group; a ureagroup; amino; and the like, including straight or branched chainderivatives thereof, cyclic derivatives thereof, substituted derivativesthereof, heteroatom derivatives thereof, heterocyclic derivativesthereof, functionalized derivatives thereof, salts thereof, such saltsincluding, but not limited to hydrochlorides and acetates, isomersthereof, or combinations thereof. For example, nitrogen-containingheterocyclic moieties include, but are not limited to, groups such aspyridinyl (derived from pyridine, and bonded through a ring carbon),piperidinyl (derived from piperidine and bonded through the ringnitrogen atom or a ring carbon), and pyrrolidinyl (derived frompyrrolidine and bonded through the ring nitrogen atom or a ring carbon).Examples of substituted, or functionalized, derivatives of R₁, R₂, andR₃ include, but are not limited to, moieties containing substituentssuch as acyl, formyl, hydroxy, acyl halide, amide, amino, azido, acid,alkoxy, aryloxy, halide, carbonyl, ether, ester, thioether, thioester,nitrile, alkylthio, arythio, sulfonic acid and salts thereof, thiol,alkenyl, alkynyl, nitro, imine, imide, alkyl, aryl, combinationsthereof, and the like. Moreover, in the case of alkylated derivatives ofthe recited moieties, the alkyl substituent may be pendant to therecited chemical moiety, or used for bonding to the amine nitrogenthrough the alkyl substituent.

Examples of chemical moieties R₁, R₂, and R₃ of the present inventioninclude, but are not limited to: H; methyl; ethyl; propyl; butyl;pentyl; hexyl; heptyl; octyl; ethenyl; propenyl; butenyl; ethynyl;propynyl; butynyl; cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl;cyclooctyl cyclobutenyl; cyclopentenyl; cyclohexenyl; phenyl; tolyl;xylyl; benzyl; naphthyl; pyridinyl; furanyl; tetrahydro-1-napthyl;piperidinyl; indolyl; indolinyl; pyrrolidinyl;2-(methoxymethyl)pyrrolidinyl; piperazinyl; quinolinyl; quinolyl;alkylated-1,3-dioxolane; triazinyl; morpholinyl; phenyl pyrazolyl;indanyl; indonyl; pyrazolyl; thiadiazolyl; rhodaninyl; thiolactonyl;dibenzofuranyl; benzothiazolyl; homopiperidinyl; thiazolyl;quinonuclidinyl; isoxazolidinonyl; any isomers, derivatives, orsubstituted analogs thereof; or any substituted or unsubstitutedchemical species such as alcohol, ether, thiol, thioether, tertiaryamine, secondary amine, primary amine, ester, thioester, carboxylicacid, diol, diester, acrylic acid, acrylic ester, methionine ethylester, benzyl-1-cysteine ethyl ester, imine, aldehyde, ketone, amide, ordiene. Further examples of chemical moieties R₁, R₂, and R₃ of thepresent invention include, but are not limited to, the following speciesor substituted or alkylated derivatives of the following species,covalently bonded to the amine nitrogen: furan; tetrahydrofuran; indole;piperazine; pyrrolidine; pyrrolidinone; pyridine; quinoline; anthracene;tetrahydroquinoline; naphthalene; pyrazole; imidazolc; thiophene;pyrrolidine; morpholine; and the like. One feature of the recitedspecies or substituted or alkylated derivatives of these species, isthat they may be covalently bonded to the amine nitrogen in any fashion,including through the pendant substituent or alkyl group, through theheteroatom as appropriate, or through a ring atom as appropriate, asunderstood by one of ordinary skill in the art.

The chemical moieties R₁, R₂, and R₃ of the present invention alsoinclude, but are not limited to, cyclic alkanes and cyclic alkenes, andinclude bridged and non-bridged rings. Examples of bridged ringsinclude, but are not limited to, the following groups: isopinocamphenyl;bornyl; norbornyl; adamantanetetyl; cis-(−)myrtanyl; adamantyl;noradamantyl; 6-azabicyclo[3.2.1]octane; exo-norbornane; and the like.

In one embodiment of the present invention, NR₂R₃ is derived from acyclic secondary amine. Examples of a cyclic chemical moiety, NR₂R₃, ofthe present invention include, but are not limited to,4-benzyl-piperidine; 3-piperidinemethanol; piperidine; tryptamine;moropholine; 4-piperidinopiperidine; ethyl 1-piperazine carboxylate;1-(2-amino-ethyl)-piperazine; decahydroquinoline;1,2,3,4-tetrahydro-pyridoindole (reaction at either amine);3-amino-5-phenyl pyrazole; 3-aminopyrazole;1-(2-fluorophenyl)piperazine; 1-proline methyl ester; histidinol;1-piperonyl-piperazine; hexamethyleimine; 4-hydroxypiperidine;2-piperidinemethanol; 1,3,3-trimethyl-6-azabicyclo[3.2.1]octane;3-pyrrolidinol; 1-methylpiperazine;(S)-(+)-(2-pyrrolidinylmethyl)pyrrolidine; 1-methylhomopiperazine;2-ethyl-piperidine; 1,2,3,4-tetrahydroisoquinoline;1-(4-fluorophenyl)piperazine; d,l-tryptophan methyl ester; tert-butyl(15, 45)-(−)-2,5-diazabiclyclo[2.2.1]heptane-2-carboxylate;isonipecotamide; heptamethyleneimine; alpha-methyltryptamine;6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline; 3-aminopyrrolidine;3,5-dimethylpiperidine; 2,6-dimethylmorpholine;1,4-dioxo-8-azaspiro[4.5]decane;1-methol-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline;1,3,4,6,7,8-hexahydro-2H-pyrido(1,2-A)pyrimidine;1,2,3,4-tetrahydroquinoline; 1-(2-methoxyphenyl)piperazine;1-(2-(2-hydroxyethoxy)ethyl)piperazine;(S)-(+)-2-(aminomethyl)pyrroli-dine; (3S(3a, 4Ab),8Ab)-N-t-butyl-D-ecahydro-3-isoquino-linecarboxamide; (R)-cycloserine;homopiperazine; 2,6-dimethylpiperazine (reaction at either amine);iminodibenzyl; 5-methoxytryptamine; 4,4′-bipiperidine;1-(2-hydroxyethyl)piperazine; 4-methylpiperidine; 1-histidine methylester; or methyl pipecoliate.

The R₁HN substituent is derived from a primary amine. The R₂R₃Nsubstituent is typically derived from a primary or secondary amine, butmay also arise from an amino acid, or an amino acid precursor. The aminoacid can transform into an amino alcohol. When an amino acid is employedas the source of the R₂R₃N moiety, the precursor compound may beselected from, among others, the following compounds and theirderivatives: d,l-tryptophan methyl ester; 1-methionine ethyl ester;1-lysine methyl ester (via reaction at either primary amine);(S)-benzyl-1-cysteine ethyl ester; 1-arginine methyl ester (via reactionat either primary amine); 1-glutamic acid ethyl ester; 1-histidinemethyl ester; or (3S (3a, 4Ab), 8A b)-N-t-butyl-D-ecahydro-3-iso-quinolinecarboxamide.

The R₄ moiety of the substituted ethylene diamine compounds of thepresent invention is typically selected from H, alkyl or aryl, but R₄can also constitute alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl,cycloalkyl, cycloalkenyl, and the like. Examples of the R₄ chemicalmoiety include, but are not limited to: H; methyl; ethyl; propyl; butyl;pentyl; hexyl, heptyl; octyl; ethenyl; propenyl; butenyl; ethynyl;propynyl; butynyl; cyclobutyl; cyclopentyl; cyclohexyl; cyclobutenyl;cyclopentenyl; cyclohexenyl; phenyl; tolyl; xylyl; benzyl; naphthyl;straight or branched chain derivatives thereof; cyclic derivativesthereof; substituted, functionalized, and heteroatom derivativesthereof; and heterocyclic derivatives thereof, and the like. Typically,R₄ is selected from H, methyl, ethyl, butyl or phenyl. However, when R₄is “H” the ethylene diamine does not contain ethambutol.

A majority of the ethylene diamine compounds described herein arepreferably prepared using a solid support synthesis, as set forth in oneof the representative reaction schemes shown in FIG. 1. However, when R₄is H, the reaction does not proceed well when sterically hindered aminesare used for R₁NH₂, or when diamines, such as amino alkylenemorpholine,or aminoalkylene-piperidines, are used for R₁NH₂. When R₄ is methyl, orphenyl, sterically hindered amines used for R₃R₂NH do not work well dueto steric hindrance at the reaction site. In this case, a competinghydrolysis reaction producing the corresponding amino alcohols, andincomplete reduction of the amidoethyleneamines, interfere with thereaction scheme. As a result, the desired diamine products form in lowyields.

The preparation of the ethylene diamines is preferably accomplished insix steps, using a rink-acid resin. The first step of the synthesis isconverting the rink-acid resin to rink-chloride by treatment withtriphenylphosphine and hexachloroethane in tetrahydrofuran (THF). Thisstep is followed by addition of the primary amine in the presence ofHunig's base (EtN(i-Pr)₂) in dichloroethane. The third step is theacylation of the resin-attached amine using either one of the twoacylation routes shown in FIG. 1. The acylation step is preferablyaccomplished using either α-chloroacetyl chloride, α-bromo-α-methylacetylbromide, α-bromo-α-ethylacetyl bromide, α-bromo-α-butylacetylbromide, or α-chloro-α-phenyl-acetylchloride, each in the presenceof pyridine in THF. Other acylation reagents known to those skilled inthe art may also be used, however, the α-bromoacetyl halides result inlow product yields, which may be attributed to HBr elimination. Theacylation may also be accomplished via a peptide coupling mechanismusing α-bromo-α-methylacetic acid, or α-chloro-α-methylacetic acid, inthe presence of benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBrop) and N₁N-diisopropylethyl amine (EtN(i-Pr)₂)in dichloromethane (DCM) and dimethylformamide (DMF). Again, otheracylation reagents known to those skilled in the art may also be used.The acylation step is preferably performed twice to achieve betteracylated product yields.

Introduction of the second nitrogen moiety is preferably achieved in thepresence of Hunig's base in dimethylformamide (DMF). Reduction of theintermediate amine-amide is carried out using Red-Al (3.4M solution ofsodium his (2-methoxyethoxy) aluminum hydride in toluene). The finalproduct is cleaved from the resin support using a 10% solution (byvolume) of trifluoroacetic acid (TFA) in dichloromethane (DCM). Thesolvent is evaporated, and the TFA salts of the final diamine productsare analyzed by mass spec, and screened against M. tuberculosis foreffectiveness. Some of the substituted ethylene diamines, prepared usingthe above-described solid-support synthesis, are also prepared using asolution phase synthesis described below.

Formation of the Substituted Ethylene Diamine Library

The solid support syntheses, shown in FIG. 1, are preferably used toprepare a substituted ethylene diamine library. Solid phase synthesisoffers at least three principal advantages: (i) a reduced need forchromatographic procedures, (ii) the use of excess reagents to drive areaction forward in high yields, and (iii) the use of split and pooltechnologies for the synthesis of a large number of compounds. Solidsupport syntheses of 1,2-diamine libraries have previously beenaccomplished by the reduction of short peptides (Cuervo et al., Peptides1994: Proceedings of the European Peptide Symposium; Maia HSL Ed., Esom:Leiden, 1995, 465-466). However, as described herein, an ethylenediamine library is created using amines, rather than simple amino acids,to allow for greater diversity in the building-block monomers. The firstthree steps of each support synthesis: the activation of the Rink-acidresin, the addition of the first amine, and the acylation step arecarried out in 10 ml tubes on a QUEST® 210 Synthesizer manufactured byARGONAUT TECHNOLOGIES®, Inc., Foster City, Calif. The synthesizerhandles up to twenty simultaneous reactions in 5 ml or 10 ml reactionvessels to allow for rapid synthesis of target compounds. Thesynthesizer provides programmable temperature control and agitation, andthe automated delivery of solvents into the reaction vessels. Theaddition of the second amine, the reduction with Red-Al, and thecleavage from the solid support are carried out in 2 ml wells in a96-well, chemically resistant plate.

Prior to the solid support synthesis, each amine, within numbers 1 to288, as shown in FIGS. 2, 3, and 4, is dissolved in DMF as a one molarsolution, and organized in three, 96-well plates (one amine per well),to yield three master plates of these amines. An individual haloacetylamide from each primary amine and a particular R₄ group, is formed inthe first three steps of the support synthesis. Individual haloacetylamides are then pooled into groups of ten or thirty. A suspension of thepooled resins in a 2:1 mixture of DCM/THF is evenly distributed intoone, two or three reaction plates to assure 15-20 mg of the suspensionper well. The number of reaction plates used is based on the amount ofsuspension available. Each well of pooled resins is reacted with acorresponding amine from the master plates. FIG. 5 provides a flowschematic for a representative pool. Each reaction occurs in a separatewell, in the presence of Hunig's base in DMF at 70-75° C. for 16-20hours. Each resulting amine-amide is reduced using 65+w % Red-Al at roomtemperature. The reduction is followed by cleavage with 10% vol. TFA inDCM. The solvents in each reaction well are evaporated, and the TFAsalts of the diamines analyzed (mass spec), and screened against M.tuberculosis. One plate of pooled diamines are screened against M.smegmatis. Two randomly selected rows in each plate; i.e., 24 samplesper 96-well plate, or 25% of the library, are examined by massspectroscopy. Specific protocols and detailed methods are provided belowin the Examples.

Screening Against M. tuberculosis

An entire library of synthesized substituted ethylene diamines (targetednumber of compounds about 100,000), prepared as described above, wasscreened, in vitro, against M. tuberculosis in ethambutol (EMB)sensitive Luc-assay. The MIC (Minimum Inhibition Concentration) was alsodetermined. The MIC is the minimum concentration of a growth inhibitor,here the substituted ethylene diamine, where there is no multiplicationof the microorganism under examination. Screening was done using aHigh-Throughout Screening (HTS) Luc assay with recombinant mycobacteriacontaining a promoter fusion of a luciferase to the EB-inducible gene(Luc assay). The Luc-assay and MIC assay are described in detail below.These assays are well known to those skilled in the art. Based on thisinitial screening, 300+ compound mixtures showed anti-TB activity. FIG.6 represents typical assay data in a luciferase reporter straincontaining an Rv0341 EMB-inducible promoter. FIG. 6 represents percentmaximum Luminescence Count per Second (% Max. LCPS) for pooled compoundmixtures in one row (row D) in one of the 96-well plates.

Deconvolution of the Reactive Wells

The M. tuberculosis screening revealed approximately 300 activecompounds mixtures that were selected for deconvolution. In particular,wells possessing activity of approximately <12.5 μM in the HTS Lucassay, and/or an MIC of approximately <12.5 μM, were selected for atotal of 336 wells.

Deconvolutions were performed by discrete re-synthesis of eachsubstituted ethylene diamine compound in each active compound pool. Thepooled compounds in each active well were individually synthesized, andscreened. Syntheses of the targeted diamine compounds in each activepool were done in the 96-well plates using stored archived α-haloacetylamides (resin attached haloacetyl amides), according to the previouslydescribed reaction steps (the addition of the second amine, thereduction with Red-Al, and the cleavage from the solid support). Thearchived resins were stored as individual compounds at 4° C. The 96-wellplates were used for the remaining synthesis steps as previouslydescribed.

The same screening tests, MIC and HTS Luc assay, were performed on eachdeconvoluted compound. Representative Luminescence data for deconvolutedcompounds are shown in FIGS. 7 and 8. FIGS. 7 and 8 represent theLuminescence Count per Second (LCPS) for individual compounds.

Summary of Screening Results

Overall, the deconvolution screening results revealed about 2,000ethylene diamine compounds with inhibitory activity against M.tuberculosis. More than 150 of these compounds exhibited MICs equal toor lower than approximately 12.5 μM. FIG. 9 summarizes the MIC data forall synthesized discrete compounds with an MIC of 50 μM or less. FIG. 10summarizes Luc assay data for all compounds that exhibit at least 10%activity at each concentration (the results are not cumulative). The MICand Luc activities were obtained for non-purified samples, with chemicalyields of approximately 20%, based on an assumed 80% yield at eachreaction step. In the Luc assay, 32 compounds exhibited activity at 1.56μM, and in the MIC assay, at least 11 compounds had an MIC of 3.13 μM.

The total frequency of the top thirteen amines that contributed to theactivity of the substituted ethylene diamines are shown in FIG. 11, witheach amine represented by its numerical designation. These aminesinclude the following:

#11 2,3-Dimethylcylochexy amines

#18 3,3-Diphenylpropylamine

#44 1-Adamantanemethylamine

#47 2,2-Diphenylethylaminne

#63 (S)-2-Amino-1-butanol

#74.1 (−)-cis-Myrtanylamine

#77.1 Cyclooctylamine

#78.1 2-Adamantamine

#105a (1R,2R,3R,5S)-(−)-Isopinocampheylamine

#231 2-Methoxyphenethylamine

#255 (S)-Cylcohexylethylamine

#266 Undecylamine

#272 Geranylamine

Other amines that contributed to the activity of the substitutedethylene diamines are shown in Table 2. The compounds in Table 2 aresorted by their MIC results. Some compounds, synthesized in largerquantities (2-60 mg) on the Quest® Synthesizer, and purified by HPLCusing semi-preparative C18-column, are shown in Table 3. Generally, thefinal purity of each compound in Table 3 was at least 90%.

In one embodiment, the present invention comprises a compositioncomprising compound 109 and compound 73.

In another embodiment, the present invention comprises a compositioncomprising compound 109 and compound 73 and a standard tuberculosisdrug.

In yet another embodiment, the present invention comprises a method oftreating disease caused by an infectious agent comprising administeringan effective amount of compound 109.

Still a further embodiment, the present invention comprises a method oftreating disease caused by an infectious agent comprising administeringan effective amount of compound 109 and compound 73.

In yet another embodiment, the present invention comprises a method oftreating disease caused by an infectious agent comprising administeringan effective amount of compound 109 and compound 73 and a standardtuberculosis drug. In another embodiment, the present inventioncomprises a method of treating disease caused by an infectious agentcomprising administering an effective amount of compound 109 andcompound 73 and a pharmaceutical carrier.

TABLE 2 Synthetic Substituted Diethylene Diamines Sorted by MinimumInhibition Concentration N1 N2 R4 MIC (uM) % Induction3,3-Diphenylpropylamine exo-Aminonorbornane Hydrogen 3.13 53.702,2-Diphenylamine (+)-Isopinocampheylamine Hydrogen 3.13 93.942,2-Diphenylamine cis-(−)-Myrtanylamine Hydrogen 3.13 64.492,2-Diphenylamine Cyclooctylamine Hydrogen 3.13 63.44 2,2-Diphenylamine3,4-Dihydroxynorephedrine Hydrogen 3.13 42.80 5-AminoquinolineCyclohexylamine Hydrogen 3.13 18.33 5-Aminoquinoline tert-OctylamineHydrogen 3.13 20.85 5-Aminoquinoline 4-Methylcyclohexylamine Hydrogen3.13 26.33 cis-(−)-Myrtanylamine (+)-Bornylamine Hydrogen 3.13 100.00cis-(−)-Myrtanylamine 1-Adamantanemethylamine Hydrogen 3.13 85.20cis-(−)-Myrtanylamine (−)-Isopinocampheylamine Hydrogen 3.13 60.941-Adamantanemethylamine tert-Octylamine Hydrogen 4.7 9.81 3,4-1-Adamantanemethylamine Hydrogen 6.25 11.45 Dimethoxyphenethylamine 3,4-Hexetidine (mixture of Hydrogen 6.25 0 Dimethoxyphenethylamine isomers)3,4- Dehydroabietylamine Hydrogen 6.25 0 Dimethoxyphenethylamine3,3-Diphenylpropylamine 1-Adamantanemethylamine Hydrogen 6.25 9.533,3-Diphenylpropylamine 2-Methylcyclohexylamine Hydrogen 6.25 50.08 (mixof cis and trans) 3,3-Diphenylpropylamine 1,3-DimethylbutylamineHydrogen 6.25 39.40 3,3-Diphenylpropylamine 1-(1-Adamantyl)ethylamine,Hydrogen 6.25 45.14 HCl 3,3-Diphenylpropylamine (S)-(−)- Hydrogen 6.2543.49 Cyclohexylethylamine 3,3-Diphenylpropylamine (R)-(−)- Hydrogen6.25 34.54 Cyclohexylethylamine 3,3-Diphenylpropylamine1-Adamantanemethylamine Methyl 6.25 16.14 Propylamine Hexetidine(mixture of Hydrogen 6.25 0 isomers) Phenethylamine Hexetidine (mixtureof Hydrogen 6.25 0 isomers) b-Methylphenethylamine Hexetidine (mixtureof Hydrogen 6.25 0 isomers) b-Methylphenethylamine Undecylamine Hydrogen6.25 0 2,2-Diphenylamine (+)-Bornylamine Hydrogen 6.25 87.862,2-Diphenylamine (−)-Isopinocampheylamine Hydrogen 6.25 77.802,2-Diphenylamine alpha-Methyltryptamine Hydrogen 6.25 55.072,2-Diphenylamine alpha-Methyltryptamine Hydrogen 6.25 23.082,2-Diphenylamine 4-Phenylbutylamine Hydrogen 6.25 2,2-Diphenylamine2,5- Hydrogen 6.25 Dimethoxyphenethylamine 2,2-Diphenylamine2,4-Dichlorophenethylamine Hydrogen 6.25 2,2-Diphenylamine2-(2-Aminomethyl) Hydrogen 6.25 phenylthio)benzyl alcohol2,2-Diphenylamine 1-(1-Naphthyl)ethylamine Hydrogen 6.25 7.20 Veratrylamine 2,5- Hydrogen 6.25 Dimethoxyphenethylamine Veratryl amine2-(2-Aminomethyl) Hydrogen 6.25 phenylthio)benzyl alcohol5-Aminoquinoline 2-Aminoheptane Hydrogen 6.25 26.22 5-Aminoquinoline1-Adamantanamine Hydrogen 6.25 18.91 1-Aminomethyl-1- Hexetidine(mixture of Hydrogen 6.25 cyclohexanol, HCl isomers)cis-(−)-Myrtanylamine 2,3- Hydrogen 6.25 100.00 Dimethylcyclohexylaminecis-(−)-Myrtanylamine 3,3-Diphenylpropylamine Hydrogen 6.25 87.78cis-(−)-Myrtanylamine (+)-Isopinocampheylamine Hydrogen 6.25 93.10cis-(−)-Myrtanylamine 2,2-Diphenylamine Hydrogen 6.25 81.84cis-(−)-Myrtanylamine cis-(−)-Myrtanylamine Hydrogen 6.25 68.24cis-(−)-Myrtanylamine 1,3,3-Trimethyl-6- Hydrogen 6.25 68.18azabicyclo[3.2.1]octane cis-(−)-Myrtanylamine 1-AdamantanemethylamineMethyl 6.25 24.22 cis-(−)-Myrtanylamine cis-(−)-Myrtanylamine Methyl6.25 44.14 Cyclooctylamine 3,3-Diphenylpropylamine Hydrogen 6.25 100.00Cyclooctylamine (−)-Isopinocampheylamine Hydrogen 6.25 59.13sec-Butylamine Hexetidine (mixture of Hydrogen 6.25 isomers)3-Methylbenzylamine Hexetidine (mixture of Hydrogen 6.25 isomers)3-Methylbenzylamine Undecylamine Hydrogen 6.25 2-MethoxyethylamineHexetidine (mixture of Hydrogen 6.25 isomers) Geranylamine2-Adamantanamine, HCl Hydrogen 6.25 25.66 1-Adamantanemethylamine4-Benzylpiperidine Hydrogen 9.4 0 1-Adamantanemethylamine 2,3- Hydrogen9.4 0 Dimethylcyclohexylamine 1-Adamantanemethylamine3,3-Diphenylpropylamine Hydrogen 9.4 40.06 1-Adamantanemethylamine1-Adamantanemethylamine Hydrogen 9.4 15.25 1-Adamantanemethylamine2,2-Diphenylamine Hydrogen 9.4 0 1-Adamantanemethylamine1,3,3-Trimethyl-6- Hydrogen 9.4 0 azabicyclo[3.2.1]octane1-Adamantanemethylamine 138 Hydrogen 9.4 0 3-Phenyl-1-propylamine 138Hydrogen 9.4 2,2-Diphenylamine 1-Adamantanemethylamine Hydrogen 9.465.89 2,2-Diphenylamine 138 Hydrogen 9.4 Furfurylamine Hexetidine(mixture of Hydrogen 12.5 0 isomers) 3,4,5- Hexetidine (mixture ofHydrogen 12.5 0 Trimethoxybenzylamine isomers) 1-Methyl-3-Dehydroabietylamine Hydrogen 12.5 0 phenylpropylamine CyclobutylamineHexetidine (mixture of Hydrogen 12.5 0 isomers) 2-FluorobenzylamineHexetidine (mixture of Hydrogen 12.5 0 isomers) 2-FluorobenzylamineDehydroabietylamine Hydrogen 12.5 0 3,4- Undecylamine Hydrogen 12.5 0Dimethoxyphenethylamine 3,3-Diphenylpropylamine exo-AminonorbornaneHydrogen 12.5 14.38 3,3-Diphenylpropylamine Decahydroquinoline Hydrogen12.5 22.52 3,3-Diphenylpropylamine Hexetidine (mixture of Hydrogen 12.50 isomers) 3,3-Diphenylpropylamine 4-Phenylbutylamine Hydrogen 12.5 03,3-Diphenylpropylamine 2-Methoxyphenethylamine Hydrogen 12.5 6.823,3-Diphenylpropylamine 2,4-Dichlorophenethylamine Hydrogen 12.5 03,3-Diphenylpropylamine 1-Aminoindan Hydrogen 12.5 18.053,3-Diphenylpropylamine Undecylamine Hydrogen 12.5 03,3-Diphenylpropylamine Dehydroabietylamine Hydrogen 12.5 03,3-Diphenylpropylamine 2-(1- Methyl 12.5 9.5 Cyclohexenyl)ethylamine3,3-Diphenylpropylamine cis-(−)-Myrtanylamine Methyl 12.5 18.413,3-Diphenylpropylamine Cyclooctylamine Methyl 12.5 20.84 PropylamineDehydroabietylamine Hydrogen 12.5 0 Phenethylamine DehydroabietylamineHydrogen 12.5 0 Cyclohexylamine Hexetidine (mixture of Hydrogen 12.5 0isomers) 3-Amino-1-propanol Hexetidine (mixture of Hydrogen 12.5 0isomers) b-Methylphenethylamine Dehydroabietylamine Hydrogen 12.5 04-Methoxyphenethylamine 2-Fluorophenethylamine Hydrogen 12.5 04-Methoxyphenethylamine 2-(1- Hydrogen 12.5 0 Cyclohexenyl)ethylamine4-Methoxyphenethylamine 2,4-Dimethoxybenzylamine Hydrogen 12.5 04-Methoxyphenethylamine 4-Fluorophenethylamine Hydrogen 12.5 16.784-Methoxyphenethylamine Hexetidine (mixture of Hydrogen 12.5 0 isomers)Tetrahydrofurfurylamine Hexetidine (mixture of Hydrogen 12.5 0 isomers)Amylamine 4-Fluorophenethylamine Hydrogen 12.5 0 3-Phenyl-1-propylamine2-(1- Hydrogen 12.5 Cyclohexenyl)ethylamine 3-Phenyl-1-propylamine4-Fluorophenethylamine Hydrogen 12.5 12.94 2,2-Diphenylaminetert-Amylamine Hydrogen 12.5 9.05 2,2-Diphenylamine UndecylamineHydrogen 12.5 2,2-Diphenylamine Dehydroabietylamine Hydrogen 12.52,2-Diphenylamine cis-(−)-Myrtanylamine Methyl 12.5 45.181-(3-Aminopropyl)-2- 2,5- Hydrogen 12.5 pyrrolidinone (tech)Dimethoxyphenethylamine 1-(3-Aminopropyl)-2- 2-(2- Hydrogen 12.5pyrrolidinone (tech) Aminomethyl)phenylthio)benzyl alcohol 4- 2,5-Hydrogen 12.5 (Trifluoromethyl)benzylamine Dimethoxyphenethylamine 4-1-(1-Naphthyl)ethylamine Hydrogen 12.5 (Trifluoromethyl)benzylamineHydrogen 12.5 Veratryl amine 4-Phenylbutylamine Hydrogen 12.55-Amino-1-pentanol 2,5- Hydrogen 12.5 Dimethoxyphenethylamine5-Amino-1-pentanol 2-(2- Hydrogen 12.5 Aminomethyl)phenylthio)benzylalcohol 2-(1- 2-(1- Hydrogen 12.5 Cyclohexenyl)ethylamineCyclohexenyl)ethylamine 2-(1- 4-Fluorophenethylamine Hydrogen 12.5Cyclohexenyl)ethylamine 2-(1- 4-Phenylbutylamine Hydrogen 12.5Cyclohexenyl)ethylamine 2-(1- 2,5- Hydrogen 12.5 Cyclohexenyl)ethylamineDimethoxyphenethylamine 2-(1- 2-(2-Aminomethyl) Hydrogen 12.5Cyclohexenyl)ethylamine phenylthio)benzyl alcohol 1-Aminomethyl-1- 2,5-Hydrogen 12.5 cyclohexanol, HCl Dimethoxyphenethylamine3-Fluorobenzylamine 2,5- Hydrogen 12.5 Dimethoxyphenethylamine4-Amino-1-butanol Hexetidine (mixture of Hydrogen 12.5 isomers)2-Ethoxybenzylamine Hexetidine (mixture of Hydrogen 12.5 isomers)cis-(−)-Myrtanylamine Cyclooctylamine Hydrogen 12.5 67.73cis-(−)-Myrtanylamine 4-Methylcyclohexylamine Hydrogen 12.5 18.39cis-(−)-Myrtanylamine 1-Adamantanamine Hydrogen 12.5 60.16cis-(−)-Myrtanylamine 3,3-Diphenylpropylamine Methyl 12.5 22.32Cyclooctylamine (+)-Isopinocampheylamine Hydrogen 12.5 57.83Cyclooctylamine (+)-Bornylamine Hydrogen 12.5 100.00 Cyclooctylamine1-Adamantanemethylamine Hydrogen 12.5 52.95 Cyclooctylamine2,2-Diphenylamine Hydrogen 12.5 71.43 Cyclooctylaminecis-(−)-Myrtanylamine Hydrogen 12.5 84.56 CyclooctylamineCyclooctylamine Hydrogen 12.5 59.21 Cyclooctylamine Hexetidine (mixtureof Hydrogen 12.5 isomers) Cyclooctylamine Aminodiphenylmethane Hydrogen12.5 Cyclooctylamine Undecylamine Hydrogen 12.5 5.61 Cyclooctylamine3,3-Diphenylpropylamine Methyl 12.5 53.92 Cyclooctylamine(+)-Isopinocampheylamine Methyl 12.5 Cyclooctylaminecis-(−)-Myrtanylamine Methyl 12.5 33.89 4-ChlorophenylalaninolHexetidine (mixture of Hydrogen 12.5 isomers) (−)-Isopinocampheylamine3,3-Diphenylpropylamine Hydrogen 12.5 23.68 (−)-Isopinocampheylamine(+)-Bornylamine Hydrogen 12.5 44.85 (−)-Isopinocampheylamine2-Amino-1-propanol, d,l Hydrogen 12.5 46.19 (−)-Isopinocampheylaminecis-(−)-Myrtanylamine Hydrogen 12.5 33.87 (−)-Isopinocampheylamine2-Adamantanamine, HCl Hydrogen 12.5 24.29 (−)-IsopinocampheylamineAminodiphenylmethane Hydrogen 12.5 48.35 Allylamine Hexetidine (mixtureof Hydrogen 12.5 isomers) 3-Ethoxypropylamine Hexetidine (mixture ofHydrogen 12.5 isomers) sec-Butylamine Dehydroabietylamine Hydrogen 12.52-Aminoheptane Dehydroabietylamine Hydrogen 12.5 Ethanolamine Hexetidine(mixture of Hydrogen 12.5 isomers) 3-Methylbenzylamine4-Phenylbutylamine Hydrogen 12.5 3-Methylbenzylamine2,4-Dichlorophenethylamine Hydrogen 12.5 3-MethylbenzylamineDehydroabietylamine Hydrogen 12.5 Piperonylamine Hexetidine (mixture ofHydrogen 12.5 isomers) Piperonylamine Dehydroabietylamine Hydrogen 12.52-Methoxyethylamine Dehydroabietylamine Hydrogen 12.54-Fluorophenethylamine Hexetidine (mixture of Hydrogen 12.5 isomers)3-o-Methyldopamine, HCl Hexetidine (mixture of Hydrogen 12.5 isomers)3-o-Methyldopamine, HCl Undecylamine Hydrogen 12.5 3-o-Methyldopamine,HCl Dehydroabietylamine Hydrogen 12.5 3-Fluorophenethylamine Hexetidine(mixture of Hydrogen 12.5 isomers) 3-FluorophenethylamineDehydroabietylamine Hydrogen 12.5 2-Methoxyphenethylamine Hexetidine(mixture of Hydrogen 12.5 isomers) 2-MethoxyphenethylamineAminodiphenylmethane Hydrogen 12.5 34.67 2-Fluoroethylamine, HClHexetidine (mixture of Hydrogen 12.5 isomers) 2-Amino-1-phenylethanolHexetidine (mixture of Hydrogen 12.5 isomers) 2-Amino-1-phenylethanolDehydroabietylamine Hydrogen 12.5 2,5- 2-Adamantanamine, HCl Hydrogen12.5 22.18 Dimethoxyphenethylamine 2-(2- N-AllylcyclopentylamineHydrogen 12.5 62.31 Chlorophenyl)ethylamine 2-(2- Hexetidine (mixture ofHydrogen 12.5 Chlorophenyl)ethylamine isomers) 3-HydroxytyramineHexetidine (mixture of Hydrogen 12.5 isomers) 4- 2-Adamantanamine, HClHydrogen 12.5 28.34 (Trifluoromethoxy)benzylamine Geranylamine(+)-Bornylamine Hydrogen 12.5 Geranylamine 1,3,3-Trimethyl-6- Hydrogen12.5 37.42 azabicyclo[3.2.1]octane Geranylamine 2-EthylpiperidineHydrogen 12.5 29.81 Geranylamine 1-Adamantanamine Hydrogen 12.5 16.63Geranylamine N-Allylcyclopentylamine Hydrogen 12.5 74.86 GeranylamineAminodiphenylmethane Hydrogen 12.5 57.93 GeranylamineDehydroabietylamine Hydrogen 12.5 1-AdamantanemethylamineDecahydroquinoline Hydrogen 18.8 0 1-Adamantanemethylamine1-Adamantanamine Hydrogen 18.8 0 2,2-Diphenylamine 2,3- Hydrogen 18.823.60 Dimethylcyclohexylamine 2,2-Diphenylamine tert-Octylamine Hydrogen18.8 19.29 2,2-Diphenylamine Decahydroquinoline Hydrogen 18.8 8.964-Methylbenzylamine Furfurylamine Hydrogen 25 13.46 4-MethylbenzylamineBenzylamine Hydrogen 25 17.07 4-Methylbenzylamine Hexetidine (mixture ofHydrogen 25 0 isomers) 4-Methylbenzylamine Dehydroabietylamine Hydrogen25 0 Cyclopentylamine Hexetidine (mixture of Hydrogen 25 0 isomers)Cyclopentylamine Dehydroabietylamine Hydrogen 25 0 FurfurylamineFurfurylamine Hydrogen 25 0 1-Methyl-3- Hexetidine (mixture of Hydrogen25 0 phenylpropylamine isomers) 1-Methyl-3- Undecylamine Hydrogen 25 0phenylpropylamine 1,2,3,4-Tetrahydro-1- Undecylamine Hydrogen 25 6.24naphthylamine 1,2,3,4-Tetrahydro-1- Dehydroabietylamine Hydrogen 25 0naphthylamine 2,3- Undecylamine Hydrogen 25 0 Dimethylcyclohexylamine2,3- Dehydroabietylamine Hydrogen 25 0 Dimethylcyclohexylamine TyramineHexetidine (mixture of Hydrogen 25 0 isomers) Tyramine UndecylamineHydrogen 25 0 Tyramine Dehydroabietylamine Hydrogen 25 0 Tyraminecis-(−)-Myrtanylamine Methyl 25 0 2-Fluorobenzylamine UndecylamineHydrogen 25 0 (R)-2-Amino-1-butanol Hexetidine (mixture of Hydrogen 25 0isomers) 3,3-Diphenylpropylamine (S)-(+)-1-Amino-2-propanol Hydrogen 250 3,3-Diphenylpropylamine 2-Ethylpiperidine Hydrogen 25 11.323,3-Diphenylpropylamine N-Allylcyclopentylamine Hydrogen 25 11.633,3-Diphenylpropylamine Aminodiphenylmethane Hydrogen 25 03,3-Diphenylpropylamine 3,5-Dimethylpiperidine (cis- Hydrogen 25 30.28and trans-) 3,3-Diphenylpropylamine Allylcyclohexylamine Hydrogen 259.10 Propylamine Undecylamine Hydrogen 25 0 Phenethylamine UndecylamineHydrogen 25 0 Tryptamine (S)-(+)-1-Amino-2-propanol Hydrogen 25 0Tryptamine 2-Amino-2-methyl-1- Hydrogen 25 0 propanol CyclohexylamineUndecylamine Hydrogen 25 0 Cyclohexylamine Dehydroabietylamine Hydrogen25 0 (+)-Isopinocampheylamine Dehydroabietylamine Hydrogen 25 0Benzylamine Hexetidine (mixture of Hydrogen 25 isomers) BenzylamineUndecylamine Hydrogen 25 3-Amino-1-propanol Dehydroabietylamine Hydrogen25 0 2-Fluorophenethylamine 2-Fluorophenethylamine Hydrogen 25 02-Fluorophenethylamine Veratryl amine Hydrogen 25 02-Fluorophenethylamine 2,4-Dimethoxybenzylamine Hydrogen 25 02-Fluorophenethylamine 2-Amino-2-methyl-1- Hydrogen 25 0 propanol2-Fluorophenethylamine 4-Fluorophenethylamine Hydrogen 25 02-Fluorophenethylamine Hexetidine (mixture of Hydrogen 25 0 isomers)2-Fluorophenethylamine 1-(1-Naphthyl)ethylamine Hydrogen 25 02-Fluorophenethylamine 1-Adamantanemethylamine Methyl 25 3.212-Fluorophenethylamine cis-(−)-Myrtanylamine Methyl 25 4.89b-Methylphenethylamine 4-Phenylbutylamine Hydrogen 25 0b-Methylphenethylamine 2,4-Dichlorophenethylamine Hydrogen 25 0b-Methylphenethylamine 1-(1-Naphthyl)ethylamine Hydrogen 25 04-Methoxyphenethylamine 1-Adamantanemethylamine Hydrogen 25 04-Methoxyphenethylamine 1-(3-Aminopropyl)-2- Hydrogen 25 0 pyrrolidinone(tech) 4-Methoxyphenethylamine Veratryl amine Hydrogen 25 04-Methoxyphenethylamine Undecylamine Hydrogen 25 04-Methoxyphenethylamine Dehydroabietylamine Hydrogen 25 0Tetrahydrofurfurylamine Dehydroabietylamine Hydrogen 25 0 Amylamine2-Fluorophenethylamine Hydrogen 25 0 Amylamine 2-(1- Hydrogen 25 0Cyclohexenyl)ethylamine Amylamine 2,4-Dimethoxybenzylamine Hydrogen 25 03-Phenyl-1-propylamine 2-Fluorophenethylamine Hydrogen 253-Phenyl-1-propylamine 1-Adamantanemethylamine Hydrogen 253-Phenyl-1-propylamine 2,4-Dimethoxybenzylamine Hydrogen 253-Phenyl-1-propylamine Hexetidine (mixture of Hydrogen 25 isomers)3-Phenyl-1-propylamine 4-Phenylbutylamine Hydrogen 253-Phenyl-1-propylamine 2,4-Dichlorophenethylamine Hydrogen 253-Phenyl-1-propylamine Undecylamine Hydrogen 25 3-Phenyl-1-propylamineDehydroabietylamine Hydrogen 25 2,2-Diphenylamine4-(2-Aminoethyl)morpholine Hydrogen 25 2,2-Diphenylamine1-(3-Aminopropyl)-2- Hydrogen 25 pyrrolidinone (tech) 2,2-Diphenylamine2-(1- Hydrogen 25 Cyclohexenyl)ethylamine 2,2-Diphenylamine2,4-Dimethoxybenzylamine Hydrogen 25 2,2-Diphenylamine 4-(3- Hydrogen 25Aminopropyl)morpholine 2,2-Diphenylamine 4-Fluorophenethylamine Hydrogen25 2,2-Diphenylamine Hexetidine (mixture of Hydrogen 25 isomers)2,2-Diphenylamine (S)-(−)- Hydrogen 25 Cyclohexylethylamine2,2-Diphenylamine 1-Adamantanemethylamine Methyl 25 5.841-(3-Aminopropyl)-2- 4-Phenylbutylamine Hydrogen 25 pyrrolidinone (tech)4- 1-Adamantanemethylamine Hydrogen 25 (Trifluoromethyl)benzylamine 4-tert-Amylamine Hydrogen 25 (Trifluoromethyl)benzylamine 4-alpha-Methyltryptamine Hydrogen 25 6.06 (Trifluoromethyl)benzylamine 4-4-Phenylbutylamine Hydrogen 25 (Trifluoromethyl)benzylamine 4-2-(2-Aminomethyl)phenylthio)benzyl Hydrogen 25 5.13(Trifluoromethyl)benzylamine alcohol 4- Undecylamine Hydrogen 25(Trifluoromethyl)benzylamine 4- (−)-3,4- Hydrogen 25(Trifluoromethyl)benzylamine Dihydroxynorephedrine 4-Dehydroabietylamine Hydrogen 25 (Trifluoromethyl)benzylamine Veratrylamine tert-Amylamine Hydrogen 25 5-Amino-1-pentanol 4-PhenylbutylamineHydrogen 25 2-(1- 2-Fluorophenethylamine Hydrogen 25Cyclohexenyl)ethylamine 2-(1- 1-Adamantanemethylamine Hydrogen 25Cyclohexenyl)ethylamine 1-Aminomethyl-1- 4-Phenylbutylamine Hydrogen 25cyclohexanol, HCl 3-Fluorobenzylamine 4-Phenylbutylamine Hydrogen 253-Fluorobenzylamine 2-(2- Hydrogen 25 Aminomethyl)phenylthio)benzylalcohol 2,4-Dimethoxybenzylamine 1-Adamantanamine Hydrogen 252,4-Dimethoxybenzylamine Hexetidine (mixture of Hydrogen 25 isomers)2,4-Dimethoxybenzylamine Undecylamine Hydrogen 252,4-Dimethoxybenzylamine Dehydroabietylamine Hydrogen 252-Ethoxybenzylamine 1-Adamantanamine Hydrogen 25 2-EthoxybenzylamineN-Phenylethyldiamine Hydrogen 25 2-Ethoxybenzylamine2,4-Dichlorophenethylamine Hydrogen 25 2-Ethoxybenzylamine 2-(2-Hydrogen 25 3.89 Chlorophenyl)ethylamine 2-EthoxybenzylamineUndecylamine Hydrogen 25 2-Ethoxybenzylamine DehydroabietylamineHydrogen 25 cis-(−)-Myrtanylamine 2-(1- Hydrogen 25Cyclohexenyl)ethylamine cis-(−)-Myrtanylamine Hexetidine (mixture ofHydrogen 25 isomers) cis-(−)-Myrtanylamine Aminodiphenylmethane Hydrogen25 cis-(−)-Myrtanylamine 2,4-Dichlorophenethylamine Hydrogen 25cis-(−)-Myrtanylamine (S)-(−)- Hydrogen 25 28.94 Cyclohexylethylaminecis-(−)-Myrtanylamine Undecylamine Hydrogen 25 cis-(−)-Myrtanylamine(+)-Isopinocampheylamine Methyl 25 cis-(−)-Myrtanylamine CyclooctylamineMethyl 25 24.92 Cyclooctylamine 2,3- Hydrogen 25 50.55Dimethylcyclohexylamine Cyclooctylamine (S)-2-Amino-1-butanol Hydrogen25 100.00 Cyclooctylamine 2-Adamantanamine, HCl Hydrogen 25 29.61Cyclooctylamine 4-Phenylbutylamine Hydrogen 25 Cyclooctylamine2-Chlorobenzylamine Hydrogen 25 Cyclooctylamine 2-Aminoindan, HClHydrogen 25 Cyclooctylamine Dehydroabietylamine Hydrogen 25Cyclooctylamine 1-(1-Naphthyl)ethylamine Hydrogen 25 4.62Cyclooctylamine 1-Adamantanemethylamine Methyl 25 14.202,3-Dimethoxybenzylamine Hexetidine (mixture of Hydrogen 25 isomers)2,3-Dimethoxybenzylamine Undecylamine Hydrogen 252,3-Dimethoxybenzylamine Dehydroabietylamine Hydrogen 254-Methylcyclohexylamine Hexetidine (mixture of Hydrogen 25 isomers)4-Methylcyclohexylamine Undecylamine Hydrogen 25 4-MethylcyclohexylamineDehydroabietylamine Hydrogen 25 4-Fluorobenzylamine DibenzylamineHydrogen 25 27.98 trans-2- Cyclooctylamine Hydrogen 25 32.80Phenylcyclopropylamine, HCl trans-2- 2-Adamantanamine, HCl Hydrogen 2518.99 Phenylcyclopropylamine, HCl trans-2- 1-Adamantanamine Hydrogen 2518.84 Phenylcyclopropylamine, HCl Thiomicamine Hexetidine (mixture ofHydrogen 25 isomers) (R)-1-Amino-2-propanol Hexetidine (mixture ofHydrogen 25 isomers) 4-Chlorophenylalaninol 2,4-DichlorophenethylamineHydrogen 25 4-Chlorophenylalaninol Undecylamine Hydrogen 254-Chlorophenylalaninol Dehydroabietylamine Hydrogen 25 I-LeucinolHexetidine (mixture of Hydrogen 25 isomers) I-Leucinol2,4-Dichlorophenethylamine Hydrogen 25 I-Leucinol DehydroabietylamineHydrogen 25 (−)-Isopinocampheylamine 2-Methoxyphenethylamine Hydrogen 2529.59 (−)-Isopinocampheylamine Undecylamine Hydrogen 25 AllylamineDehydroabietylamine Hydrogen 25 3-Amino-1,2-propanediol Hexetidine(mixture of Hydrogen 25 isomers) 3-Ethoxypropylamine3,3-Diphenylpropylamine Hydrogen 25 3-Ethoxypropylamine UndecylamineHydrogen 25 3-Ethoxypropylamine Dehydroabietylamine Hydrogen 25sec-Butylamine 2,4-Dichlorophenethylamine Hydrogen 25 sec-ButylamineUndecylamine Hydrogen 25 2-Aminoheptane Hexetidine (mixture of Hydrogen25 isomers) 2-Aminoheptane 4-Phenylbutylamine Hydrogen 25 2-Aminoheptane2,4-Dichlorophenethylamine Hydrogen 25 1-NaphthalenemethylamineHexetidine (mixture of Hydrogen 25 isomers) 1-Naphthalenemethylamine4-Phenylbutylamine Hydrogen 25 1-Naphthalenemethylamine2,4-Dichlorophenethylamine Hydrogen 25 1-NaphthalenemethylamineUndecylamine Hydrogen 25 Ethanolamine Dehydroabietylamine Hydrogen 25Piperonylamine 4-Phenylbutylamine Hydrogen 25 1-EthylpropylamineHexetidine (mixture of Hydrogen 25 isomers) 1-EthylpropylamineDehydroabietylamine Hydrogen 25 Isopropylamine Hexetidine (mixture ofHydrogen 25 isomers) 4-Fluorophenethylamine 4-Phenylbutylamine Hydrogen25 4-Fluorophenethylamine 2,4-Dichlorophenethylamine Hydrogen 254-Fluorophenethylamine Dehydroabietylamine Hydrogen 253-Fluorophenethylamine Undecylamine Hydrogen 25 2-Thiopheneethylamine2-Adamantanamine, HCl Hydrogen 25 19.09 2-MethylcyclohexylamineHexetidine (mixture of Hydrogen 25 (mix of cis and trans) isomers)2-Methylcyclohexylamine Dehydroabietylamine Hydrogen 25 (mix of cis andtrans) 2-Methoxyphenethylamine 2-Adamantanamine, HCl Hydrogen 25 26.772-Methoxyphenethylamine (−)-Isopinocampheylamine Hydrogen 25 31.952-Methoxyphenethylamine 1-Adamantanamine Hydrogen 25 24.382-Methoxyphenethylamine N-Allylcyclopentylamine Hydrogen 25 14.562-Methoxyphenethylamine 4-Phenylbutylamine Hydrogen 252-Methoxyphenethylamine Undecylamine Hydrogen 25 2-MethoxyphenethylamineDehydroabietylamine Hydrogen 25 2-Fluoroethylamine, HCl UndecylamineHydrogen 25 2-Fluoroethylamine, HCl Dehydroabietylamine Hydrogen 252-Aminoindan, HCl 2-Adamantanamine, HCl Hydrogen 25 17.722-Amino-1-phenylethanol Undecylamine Hydrogen 25 2,5- (+)-BornylamineHydrogen 25 25.78 Dimethoxyphenethylamine 2,5- Noradamantamine, HClHydrogen 25 11.73 Dimethoxyphenethylamine 2,5- 1-Adamantanamine Hydrogen25 12.57 Dimethoxyphenethylamine 2-(2- 4-Phenylbutylamine Hydrogen 25Chlorophenyl)ethylamine 2-(2- Undecylamine Hydrogen 25Chlorophenyl)ethylamine 2-(2- 1-(1-Naphthyl)ethylamine Hydrogen 25Chlorophenyl)ethylamine 2-(2- Hexetidine (mixture of Hydrogen 25Aminomethyl)phenylthio)benzyl isomers) alcohol 2-(2- 4-PhenylbutylamineHydrogen 25 Aminomethyl)phenylthio)benzyl alcohol 2-(2- UndecylamineHydrogen 25 Aminomethyl)phenylthio)benzyl alcohol 1-AminoindanHexetidine (mixture of Hydrogen 25 isomers) 1-Aminoindan UndecylamineHydrogen 25 1-Aminoindan Dehydroabietylamine Hydrogen 251,3-Dimethylbutylamine Hexetidine (mixture of Hydrogen 25 isomers)1,3-Dimethylbutylamine Undecylamine Hydrogen 25 5.921,3-Dimethylbutylamine Dehydroabietylamine Hydrogen 25(S)-(−)-Cyclohexylethylamine (−)-Isopinocampheylamine Hydrogen 25 19.31(S)-(−)-Cyclohexylethylamine Hexetidine (mixture of Hydrogen 25 isomers)(S)-(−)-Cyclohexylethylamine Undecylamine Hydrogen 25 10.88(S)-(−)-Cyclohexylethylamine Dehydroabietylamine Hydrogen 25(S)-(−)-2-Amino-3-phenyl-1- Hexetidine (mixture of Hydrogen 25 propanolisomers) (S)-(−)-2-Amino-3-phenyl-1- Undecylamine Hydrogen 25 propanol(S)-(−)-2-Amino-3-phenyl-1- Dehydroabietylamine Hydrogen 25 propanol(1S,2S)-(+)-2-Amino-3- Hexetidine (mixture of Hydrogen 25methoxy-1-phenyl-1- isomers) propanol Octadecylamine (+)-BornylamineHydrogen 25 Octadecylamine 1-Adamantanamine Hydrogen 25 Geranylamine2,3- Hydrogen 25 14.53 Dimethylcyclohexylamine Geranylaminetert-Octylamine Hydrogen 25 15.22 Geranylamine 1-AdamantanemethylamineHydrogen 25 4.37 Geranylamine Decahydroquinoline Hydrogen 25 31.79Geranylamine Dibenzylamine Hydrogen 25 6.48 GeranylamineN-Butylbenzylamine Hydrogen 25 16.44 Geranylamine CyclooctylamineHydrogen 25 12.37 Geranylamine (−)-Isopinocampheylamine Hydrogen 25 8.95Geranylamine 1-(1-Adamantyl)ethylamine, Hydrogen 25 32.95 HClGeranylamine Undecylamine Hydrogen 25 Geranylamine1-(1-Naphthyl)ethylamine Hydrogen 25 Amylamine 1-Adamantanamine Hydrogen37.5 0 3-Phenyl-1-propylamine 3,3-Diphenylpropylamine Hydrogen 37.53-Phenyl-1-propylamine 2,2-Diphenylamine Hydrogen 37.53-Phenyl-1-propylamine 1-Adamantanamine Hydrogen 37.5 18.652,2-Diphenylamine 3,3-Diphenylpropylamine Hydrogen 37.52,2-Diphenylamine 2,2-Diphenylamine Hydrogen 37.5 5.56 2,2-Diphenylamine1,3,3-Trimethyl-6- Hydrogen 37.5 8.67 azabicyclo[3.2.1]octane2,2-Diphenylamine 1-Adamantanamine Hydrogen 37.5 58.10 4-tert-Octylamine Hydrogen 37.5 7.47 (Trifluoromethyl)benzylamine 4- 138Hydrogen 37.5 (Trifluoromethyl)benzylamine 4-Methylbenzylamine2-Fluorobenzylamine Hydrogen 50 22.10 4-Methylbenzylamine4-Fluorobenzylamine Hydrogen 50 14.62 4-Methylbenzylaminealpha-Methyltryptamine Hydrogen 50 0 4-Methylbenzylamine UndecylamineHydrogen 50 0 Cyclopentylamine Undecylamine Hydrogen 50 0 Furfurylamine2-Fluorobenzylamine Hydrogen 50 0 Furfurylamine Benzylamine Hydrogen 500 Furfurylamine 4-Fluorobenzylamine Hydrogen 50 0 Furfurylaminealpha-Methyltryptamine Hydrogen 50 0 Furfurylamine Undecylamine Hydrogen50 0 Furfurylamine Dehydroabietylamine Hydrogen 50 0 FurfurylamineFurfurylamine Hydrogen 50 0 3,4,5- 2-Fluorobenzylamine Hydrogen 50 0Trimethoxybenzylamine 3,4,5- Benzylamine Hydrogen 50 0Trimethoxybenzylamine 3,4,5- alpha-Methyltryptamine Hydrogen 50 0Trimethoxybenzylamine 3,4,5- Undecylamine Hydrogen 50 0Trimethoxybenzylamine 3,4,5- Dehydroabietylamine Hydrogen 50 0Trimethoxybenzylamine 1-Methyl-3- alpha-Methyltryptamine Hydrogen 50 0phenylpropylamine 1-Methyl-3- Octadecylamine Hydrogen 50 0phenylpropylamine Cyclobutylamine Octadecylamine Hydrogen 50 0Cyclobutylamine Undecylamine Hydrogen 50 0 CyclobutylamineDehydroabietylamine Hydrogen 50 0 1,2,3,4-Tetrahydro-1- Hexetidine(mixture of Hydrogen 50 0 naphthylamine isomers) 1,2,3,4-Tetrahydro-1-Aminodiphenylmethane Hydrogen 50 4.31 naphthylamine1,2,3,4-Tetrahydro-1- alpha-Methyltryptamine Hydrogen 50 0 naphthylamine1,2,3,4-Tetrahydro-1- 2-Methoxyphenethylamine Hydrogen 50 0naphthylamine 2,3- Hexetidine (mixture of Hydrogen 50 0Dimethylcyclohexylamine isomers) 2,3- Aminodiphenylmethane Hydrogen 503.64 Dimethylcyclohexylamine 2,3- alpha-Methyltryptamine Hydrogen 50 0Dimethylcyclohexylamine Tyramine Furfurylamine Hydrogen 50 0 Tyramine2-Fluorobenzylamine Hydrogen 50 4.07 Tyramine Benzylamine Hydrogen 50 0Tyramine 2,4-Dichlorophenethylamine Hydrogen 50 0 2-FluorobenzylamineAminodiphenylmethane Hydrogen 50 0 2-Fluorobenzylamine4-Phenylbutylamine Hydrogen 50 0 2-Fluorobenzylamine2-Methoxyphenethylamine Hydrogen 50 0 2-Fluorobenzylamine2,4-Dichlorophenethylamine Hydrogen 50 0 2-Fluorobenzylamine1,3-Dimethylbutylamine Hydrogen 50 0 2-Fluorobenzylamine1-(1-Adamantyl)ethylamine, Hydrogen 50 0 HCl (R)-2-Amino-1-butanolDehydroabietylamine Hydrogen 50 0 3,4- Aminodiphenylmethane Hydrogen 500 Dimethoxyphenethylamine 3,4- 4-Phenylbutylamine Hydrogen 50 0Dimethoxyphenethylamine 3,4- 2-Methoxyphenethylamine Hydrogen 50 0Dimethoxyphenethylamine 3,4- 2,4-Dichlorophenethylamine Hydrogen 50 0Dimethoxyphenethylamine 3,4- 1,3-Dimethylbutylamine Hydrogen 50 0Dimethoxyphenethylamine 3,3-Diphenylpropylamine Piperidine Hydrogen 50 03,3-Diphenylpropylamine 2,3- Methyl 50 7.81 Dimethylcyclohexylamine3,3-Diphenylpropylamine (−)-Isopinocamphenylamine Methyl 50 13.06Propylamine (S)-(+)-1-Amino-2-propanol Hydrogen 50 0 Phenethylamine(S)-(+)-1-Amino-2-propanol Hydrogen 50 0 Phenethylamine4-Phenylbutylamine Hydrogen 50 0 Phenethylamine2,4-Dichlorophenethylamine Hydrogen 50 0 Phenethylamine1,3-Dimethylbutylamine Hydrogen 50 0 Phenethylamine1-(1-Adamantyl)ethylamine, Hydrogen 50 0 HCl Phenethylamine1-(1-Naphthyl)ethylamine Hydrogen 50 0 4-(2-Aminoethyl)morpholine2-Amino-2-methyl-1- Hydrogen 50 0 propanol Cyclohexylamine2,4-Dichlorophenethylamine Hydrogen 50 0 exo-Aminonorbornane BenzylamineHydrogen 50 0 (+)-Isopinocampheylamine Hexetidine (mixture of Hydrogen50 0 isomers) (+)-Isopinocampheylamine Aminodiphenylmethane Hydrogen 505.07 (+)-Isopinocampheylamine 4-Phenylbutylamine Hydrogen 50 0(+)-Isopinocampheylamine 2,4-Dichlorophenethylamine Hydrogen 50 0(+)-Isopinocampheylamine Undecylamine Hydrogen 50 0 Benzylamine3,3-Diphenylpropylamine Hydrogen 50 Benzylamine 2-Amino-2-methyl-1-Hydrogen 50 propanol Benzylamine 1-(1-Naphthyl)ethylamine Hydrogen 50Benzylamine 2,4-Dichlorophenethylamine Hydrogen 50 3-Amino-1-propanolUndecylamine Hydrogen 50 0 2-Fluorophenethylamine3,3-Diphenylpropylamine Hydrogen 50 0 2-Fluorophenethylamine1-Adamantanemethylamine Hydrogen 50 0 2-Fluorophenethylamine1-(3-Aminopropyl)-2- Hydrogen 50 0 pyrrolidinone (tech)2-Fluorophenethylamine Decahydroquinoline Hydrogen 50 02-Fluorophenethylamine 1-Adamantanamine Hydrogen 50 24.342-Fluorophenethylamine 2,4-Dichlorophenethylamine Hydrogen 50 02-Fluorophenethylamine Undecylamine Hydrogen 50 0 2-FluorophenethylamineDehydroabietylamine Hydrogen 50 0 2-Fluorophenethylamine 2-(1- Methyl 500 Cyclohexenyl)ethylamine 2-Fluorophenethylamine Cyclooctylamine Methyl50 5.81 b-Methylphenethylamine 3,3-Diphenylpropylamine Hydrogen 50 0b-Methylphenethylamine tert-Octylamine Hydrogen 50 0b-Methylphenethylamine 2-(1- Hydrogen 50 0 Cyclohexenyl)ethylamineb-Methylphenethylamine 2-Amino-2-methyl-1- Hydrogen 50 0 propanolb-Methylphenethylamine 4-Fluorophenethylamine Hydrogen 50 0b-Methylphenethylamine Geranylamine Hydrogen 50 0 b-Methylphenethylamine5-Methoxytryptamine Hydrogen 50 0 4-Methoxyphenethylamine3,3-Diphenylpropylamine Hydrogen 50 0 4-Methoxyphenethylamine2-Amino-2-methyl-1- Hydrogen 50 0 propanol 4-Methoxyphenethylamine2,4-Dichlorophenethylamine Hydrogen 50 0 4-Methoxyphenethylamine1-(1-Naphthyl)ethylamine Hydrogen 50 0 L-Methioninol Hexetidine (mixtureof Hydrogen 50 0 isomers) Tetrahydrofurfurylamine1-Adamantanemethylamine Hydrogen 50 0 Tetrahydrofurfurylamine 2-(1-Hydrogen 50 0 Cyclohexenyl)ethylamine Tetrahydrofurfurylamine4-Fluorophenethylamine Hydrogen 50 0 TetrahydrofurfurylamineUndecylamine Hydrogen 50 0 Amylamine 1-Adamantanemethylamine Hydrogen 500 Amylamine Hexetidine (mixture of Hydrogen 50 0 isomers) AmylamineUndecylamine Hydrogen 50 0 Amylamine Dehydroabietylamine Hydrogen 50 01-Adamantanemethylamine cis-(−)-Myrtanylamine Methyl 50 03-Phenyl-1-propylamine 4-(2-Aminoethyl)morpholine Hydrogen 503-Phenyl-1-propylamine 1-(3-Aminopropyl)-2- Hydrogen 50 pyrrolidinone(tech) 3-Phenyl-1-propylamine Veratryl amine Hydrogen 503-Phenyl-1-propylamine Aminodiphenylmethane Hydrogen 503-Phenyl-1-propylamine 2-(2- Hydrogen 50 Aminomethyl)phenylthio)benzylalcohol 2,2-Diphenylamine 2-Fluorophenethylamine Hydrogen 502,2-Diphenylamine 3,3-Diphenylpropylamine Methyl 50 2,2-Diphenylamine(+)-Isopinocampheylamine Methyl 50 2,2-Diphenylamine (+)-BornylamineMethyl 50 2,2-Diphenylamine Cyclooctylamine Methyl 50 2,2-Diphenylamine(−)-Isopinocampheylamine Methyl 50 3.81 4- 4-(2-Aminoethyl)morpholineHydrogen 50 (Trifluoromethyl)benzylamine 4- 2-(1- Hydrogen 50(Trifluoromethyl)benzylamine Cyclohexenyl)ethylamine 4- Hexetidine(mixture of Hydrogen 50 (Trifluoromethyl)benzylamine isomers) 4-2,4-Dichlorophenethylamine Hydrogen 50 (Trifluoromethyl)benzylamine 4-(S)-(−)- Hydrogen 50 (Trifluoromethyl)benzylamine CyclohexylethylamineVeratryl amine 1-Adamantanemethylamine Hydrogen 50 Veratryl amine 2-(1-Hydrogen 50 Cyclohexenyl)ethylamine Veratryl amine4-Fluorophenethylamine Hydrogen 50 Veratryl amine Hexetidine (mixture ofHydrogen 50 isomers) Veratryl amine 2,4-Dichlorophenethylamine Hydrogen50 Veratryl amine (S)-(−)- Hydrogen 50 Cyclohexylethylamine Veratrylamine Undecylamine Hydrogen 50 Veratryl amine DehydroabietylamineHydrogen 50 Veratryl amine 1-(1-Naphthyl)ethylamine Hydrogen 505-Amino-1-pentanol 1-Adamantanemethylamine Hydrogen 505-Amino-1-pentanol Dibenzylamine Hydrogen 50 5-Amino-1-pentanolcis-(−)-Myrtanylamine Hydrogen 50 12.97 2-(1- 2,4-DimethoxybenzylamineHydrogen 50 Cyclohexenyl)ethylamine 1-Aminomethyl-1- tert-AmylamineHydrogen 50 cyclohexanol, HCl 1-Aminomethyl-1- 2-(2- Hydrogen 50cyclohexanol, HCl Aminomethyl)phenylthio)benzyl alcohol 1-Aminomethyl-1-Undecylamine Hydrogen 50 cyclohexanol, HCl 1-Aminomethyl-1-1-(1-Naphthyl)ethylamine Hydrogen 50 cyclohexanol, HCl3-Fluorobenzylamine tert-Amylamine Hydrogen 50 3-FluorobenzylamineHexetidine (mixture of Hydrogen 50 isomers) 3-FluorobenzylamineUndecylamine Hydrogen 50 4-Amino-1-butanol Undecylamine Hydrogen 504-Amino-1-butanol Dehydroabietylamine Hydrogen 502,4-Dimethoxybenzylamine N-Phenylethyldiamine Hydrogen 502,4-Dimethoxybenzylamine Aminodiphenylmethane Hydrogen 502,4-Dimethoxybenzylamine 4-Phenylbutylamine Hydrogen 502,4-Dimethoxybenzylamine 2-Chlorobenzylamine Hydrogen 502,4-Dimethoxybenzylamine 2,4-Dichlorophenethylamine Hydrogen 502,4-Dimethoxybenzylamine 2-(2- Hydrogen 50 Chlorophenyl)ethylamine2,4-Dimethoxybenzylamine 4- Hydrogen 50 (Trifluoromethoxy)benzylamine2-Ethoxybenzylamine Aminodiphenylmethane Hydrogen 50 2-Ethoxybenzylamine4-Phenylbutylamine Hydrogen 50 2-Ethoxybenzylamine 2-ChlorobenzylamineHydrogen 50 2-Ethoxybenzylamine 2-Aminoindan, HCl Hydrogen 502-Ethoxybenzylamine 2,5- Hydrogen 50 Dimethoxyphenethylamine2-Ethoxybenzylamine 4- Hydrogen 50 (Trifluoromethoxy)benzylamine2-Ethoxybenzylamine 1-(1-Naphthyl)ethylamine Hydrogen 50cis-(−)-Myrtanylamine 4-(2-Aminoethyl)morpholine Hydrogen 50cis-(−)-Myrtanylamine 2-Fluorophenethylamine Hydrogen 50cis-(−)-Myrtanylamine 1-(3-Aminopropyl)-2- Hydrogen 50 pyrrolidinone(tech) cis-(−)-Myrtanylamine Veratryl amine Hydrogen 50cis-(−)-Myrtanylamine N-Butylbenzylamine Hydrogen 50cis-(−)-Myrtanylamine 2,4-Dimethoxybenzylamine Hydrogen 50cis-(−)-Myrtanylamine 1,2,3,4- Hydrogen 50 Tetrahydropyridoindolecis-(−)-Myrtanylamine 4-Phenylbutylamine Hydrogen 50cis-(−)-Myrtanylamine 2-(2- Hydrogen 50 3.91 Chlorophenyl)ethylaminecis-(−)-Myrtanylamine 1-(1-Adamantyl)ethylamine, Hydrogen 50 10.85 HClcis-(−)-Myrtanylamine (R)-(−)- Hydrogen 50 5.89 Cyclohexylethylaminecis-(−)-Myrtanylamine Dehydroabietylamine Hydrogen 50cis-(−)-Myrtanylamine 1-(1-Naphthyl)ethylamine Hydrogen 50cis-(−)-Myrtanylamine (+)-Bornylamine Methyl 50 4.04 Cyclooctylamine4-Methylcyclohexylamine Hydrogen 50 4.55 CyclooctylamineN-Phenylethyldiamine Hydrogen 50 Cyclooctylamine 4- Hydrogen 50(Hexacylamino)benzylamine Cyclooctylamine 2,5- Hydrogen 50Dimethoxyphenethylamine Cyclooctylamine 2,4-DichlorophenethylamineHydrogen 50 3.36 Cyclooctylamine 2-(2- Hydrogen 50 9.15Chlorophenyl)ethylamine Cyclooctylamine 1-(1-Adamantyl)ethylamine,Hydrogen 50 10.62 HCl Cyclooctylamine (S)-(−)- Hydrogen 50 5.85Cyclohexylethylamine Cyclooctylamine (R)-(−)- Hydrogen 50Cyclohexylethylamine Cyclooctylamine 4- Hydrogen 50 4.54(Trifluoromethoxy)benzylamine 2-Adamantanamine, HClcis-(−)-Myrtanylamine Hydrogen 50 49.73 4-MethylcyclohexylamineN-Phenylethyldiamine Hydrogen 50 4-Methylcyclohexylamine4-Phenylbutylamine Hydrogen 50 4-FluorobenzylamineN-Benzyl-2-phenethylamine Hydrogen 50 4-Fluorobenzylamine Hexetidine(mixture of Hydrogen 50 isomers) 4-Fluorobenzylamine UndecylamineHydrogen 50 4-Fluorobenzylamine Dehydroabietylamine Hydrogen 50 trans-2-Hexetidine (mixture of Hydrogen 50 Phenylcyclopropylamine, isomers) HCltrans-2- Undecylamine Hydrogen 50 Phenylcyclopropylamine, HCl trans-2-Dehydroabietylamine Hydrogen 50 Phenylcyclopropylamine, HCl(R)-1-Amino-2-propanol 4- Hydrogen 50 (Hexacylamino)benzylamine(R)-1-Amino-2-propanol Undecylamine Hydrogen 50 (R)-1-Amino-2-propanolDehydroabietylamine Hydrogen 50 I-Leucinol Undecylamine Hydrogen 50(−)-Isopinocampheylamine 2-Ethoxybenzylamine Hydrogen 50 27.27(−)-Isopinocampheylamine Hexetidine (mixture of Hydrogen 50 isomers)(−)-Isopinocampheylamine 4-Phenylbutylamine Hydrogen 50(−)-Isopinocampheylamine Dehydroabietylamine Hydrogen 50(−)-Isopinocampheylamine 1-(1-Naphthyl)ethylamine Hydrogen 50 Allylamine3,3-Diphenylpropylamine Hydrogen 50 Allylamine 2-Amino-1-propanol, d,lHydrogen 50 Allylamine Undecylamine Hydrogen 50 3-Amino-1,2-propanediolDehydroabietylamine Hydrogen 50 3-Ethoxypropylamine 2,2-DiphenylamineHydrogen 50 95.81 3-Ethoxypropylamine cis-(−)-Myrtanylamine Hydrogen 502-Aminoheptane 2-(2- Hydrogen 50 Aminomethyl)phenylthio)benzyl alcohol1-Naphthalenemethylamine Geranylamine Hydrogen 501-Naphthalenemethylamine Dehydroabietylamine Hydrogen 501-Aminopyrrolidine, HCl Hexetidine (mixture of Hydrogen 50 isomers)1-Aminopyrrolidine, HCl Undecylamine Hydrogen 50 1-Aminopyrrolidine, HClDehydroabietylamine Hydrogen 50 Ethanolamine 3,3-DiphenylpropylamineHydrogen 50 3-Methylbenzylamine Geranylamine Hydrogen 503-Methylbenzylamine 5-Methoxytryptamine Hydrogen 50 PiperonylamineAminodiphenylmethane Hydrogen 50 Piperonylamine2,4-Dichlorophenethylamine Hydrogen 50 Piperonylamine 2-(2- Hydrogen 50Aminomethyl)phenylthio)benzyl alcohol Isopropylamine DehydroabietylamineHydrogen 50 4-Fluorophenethylamine 2,4-Dimethoxybenzylamine Hydrogen 504-Fluorophenethylamine Aminodiphenylmethane Hydrogen 504-Fluorophenethylamine 2-(2- Hydrogen 50 Aminomethyl)phenylthio)benzylalcohol 4-Chloroamphetamine, HCl N-Allylcyclopentylamine Hydrogen 5010.25 4-Chloroamphetamine, HCl Hexetidine (mixture of Hydrogen 50isomers) 4-Chloroamphetamine, HCl 4-Phenylbutylamine Hydrogen 504-Chloroamphetamine, HCl 2-Methoxyphenethylamine Hydrogen 504-Chloroamphetamine, HCl Undecylamine Hydrogen 50 4-Chloroamphetamine,HCl Dehydroabietylamine Hydrogen 50 3-Fluorophenethylamine(−)-Isopinocampheylamine Hydrogen 50 3-Fluorophenethylamine1-Adamantamine Hydrogen 50 8.59 3-Fluorophenethylamine4-Phenylbutylamine Hydrogen 50 2-Methylcyclohexylamine UndecylamineHydrogen 50 (mix of cis and trans) 2-Methoxyphenethylamine3,3-Diphenylpropylamine Hydrogen 50 2-Methoxyphenethylamine(+)-Bornylamine Hydrogen 50 2-Methoxyphenethylamine tert-OctylamineHydrogen 50 20.46 2-Methoxyphenethylamine 1-AdamantanemethylamineHydrogen 50 2-Methoxyphenethylamine Dibenzylamine Hydrogen 502-Methoxyphenethylamine N-Butylbenzylamine Hydrogen 50 5.202-Methoxyphenethylamine 1,3,3-Trimethyl-6- Hydrogen 50 8.59azabicyclo[3.2.1]octane 2-Methoxyphenethylamine N-PhenylethyldiamineHydrogen 50 2-Methoxyphenethylamine 2,4-Dichlorophenethylamine Hydrogen50 2-Methoxyphenethylamine 2-(2- Hydrogen 50 Chlorophenyl)ethylamine2-Methoxyphenethylamine 1-(1-Adamantyl)ethylamine, Hydrogen 50 3.61 HCl2-Aminoindan, HCl (+)-Bornylamine Hydrogen 50 2-Aminoindan, HClNoradamantamine, HCl Hydrogen 50 7.43 2-(2- N-PhenylethyldiamineHydrogen 50 Chlorophenyl)ethylamine 2-(2- Aminodiphenylmethane Hydrogen50 Chlorophenyl)ethylamine 2-(2- 2,4-Dichlorophenethylamine Hydrogen 50Chlorophenyl)ethylamine 2-(2- 1-(1-Adamantyl)ethylamine, Hydrogen 50Chlorophenyl)ethylamine HCl 2-(2- Dehydroabietylamine Hydrogen 50Chlorophenyl)ethylamine 2-(2- 2-Methoxyphenethylamine Hydrogen 50Aminomethyl)phenylthio)benzyl alcohol 2-(2- 2,5- Hydrogen 50Aminomethyl)phenylthio)benzyl Dimethoxyphenethylamine alcohol 2-(2-2-(2- Hydrogen 50 Aminomethyl)phenylthio)benzyl Chlorophenyl)ethylaminealcohol 2-(2- 1-(1-Adamantyl)ethylamine, Hydrogen 50Aminomethyl)phenylthio)benzyl HCl alcohol 2-(2- DehydroabietylamineHydrogen 50 Aminomethyl)phenylthio)benzyl alcohol 1-Aminoindan4-Phenylbutylamine Hydrogen 50 1-Aminoindan 2,4-DichlorophenethylamineHydrogen 50 1,3-Dimethylbutylamine 4-Phenylbutylamine Hydrogen 50(S)-(−)-Cyclohexylethylamine Aminodiphenylmethane Hydrogen 50(S)-(−)-Cyclohexylethylamine 4-Phenylbutylamine Hydrogen 50(S)-(−)-Cyclohexylethylamine 2,4-Dichlorophenethylamine Hydrogen 50(S)-(−)-Cyclohexylethylamine 1-(1-Adamantyl)ethylamine, Hydrogen 50 HCl(1S,2S)-(+)-2-Amino-3- Dehydroabietylamine Hydrogen 50methoxy-1-phenyl-1- propanol Octadecylamine 2-Adamantanamine, HClHydrogen 50 3-Hydroxytyramine (1R,2S)-(−)-2-Amino-1,2- Hydrogen 50diphenylethanol 3-Hydroxytyramine Dehydroabietylamine Hydrogen 50Geranylamine 3,3-Diphenylpropylamine Hydrogen 50 GeranylamineN-Phenylethyldiamine Hydrogen 50 Geranylamine Hexetidine (mixture ofHydrogen 50 isomers) Geranylamine 2-Thiopheneethylamine Hydrogen 50Geranylamine 2-Methoxyphenethylamine Hydrogen 50 Geranylamine 2,5-Hydrogen 50 Dimethoxyphenethylamine Geranylamine2,4-Dichlorophenethylamine Hydrogen 50 Geranylamine 2-(2- Hydrogen 50Chlorophenyl)ethylamine 2-Fluorophenethylamine 2,3- Methyl >50 2.07Dimethylcyclohexylamine 4- 2,3- Hydrogen >50 8.20(Trifluoromethyl)benzylamine Dimethylcyclohexylamine 4- 1-AdamantanamineHydrogen >50 32.02 (Trifluoromethyl)benzylamine 5-Aminoquinolineexo-Aminonorbornane Hydrogen >50 17.87

TABLE 3 Compounds Synthesized in Larger Quantities for Further in vitroEvaluations Amount, Yields, Cmpd # Name Structure mg % 1N-(4-Methylphenyl)-N′- (furfuryl)ethane-1,2- diamine

23 25 2 N-(4-Methylphenyl)-N′- (benzyl)ethane-1,2-diamine

27 29 3 N-[1-(1,2,3,4-Tetrahydro- naphthalene)-N′-(undecenyl)-ethane-1,2- diamine

11 10 4 N-[2-(3,4-Dimethoxy- phenyl)-ethyl-N′-(1-methyladamantyl)-ethane- 1,2-diamine

13 11 5 N-[2-(3,4-Dimethoxy- phenyl)ethyl-N′- (norbornyl)-ethane-1,2-diamine

9 8 6 N-(1-Adamantylmethyl)- N′-(3,3- diphenylpropyl)propane-1,2-diamine

55 36 7 N-(1-Adamantylmethyl)- N′-(3,3- diphenylpropyl)ethane-1,2-diamine

28 22 8 N-[2-(Cyclohexen-1- yl)ethyl]-N′-(3,3- diphenylpropyl)-propane-1,2-diamine

46 37 10 N-(−)-cis-Myrtanyl- N′-(3,3- diphenylpropyl)ethane- 1,2-diamine

14 11 11 N-Cyclooctyl-N′-(3,3- diphenylpropyl)ethane- 1,2-diamine

22 18 13 N-Allyl-N-cyclopentyl- N′-(3,3- diphenylpropyl)ethane-1,2-diamine

33 27 14 N-(3,3-Diphenylpropyl)- N′-exo-(2- norborny)ethane-1,2- diamine

17 16 15 1-{2-[N-(3,3- Diphenylpropyl)]- aminoethyl}-3,5-dimethyl-piperidine

6.2 5 17 N-2-(2- Methoxyphenyl)ethyl-N′- (3,3- diphenylethyl)ethane-1,2-diamine

50 40 21 N-(3,3-Diphenylpropyl)- N′-(1S)-(1- ethylcyclohexane)-ethane-1,2-diamine

5 4 22 N-(3,3-Diphenylpropyl)- N′-(1R)-(1- ethylcyclohexane)-ethane-1,2-diamine

21 17 23 N-Allyl-N-cyclohexyl-N′- (3,3- diphenylpropyl)ethane-1,2-diamine

6 5 24 N-2-(2- Methoxyphenyl)ethyl-N′- (4-fluorophenylethyl)-ethane-1,2-diamine

10 9 27 N-(3Phenylpropyl)-N′- (1-adamantyl)ethane- 1,2-diamine

11 10 28 N-(3-Phenylpropyl)-N′- (4-fluorophenyl)ethane- 1,2-diamine

11 10 29 N-(2,2-Diphenylethyl)-N′- (2,3- dimethylcylcohexyl)ethane-1,2-diamine

4.5 4 31 N-(2,2-Diphenylethyl)-N′- (1S)-(1- ethylcyclohexane)-ethane-1,2-diamine

24 20 32 N-(2,2-Diphenylethyl)-N′- (R)-(+)-

58 48 33 N-(2,2-Diphenylethyl)-N′- (1,1,3,3- tetramethylbutyl)-ethane-1,2-diamine

11 9 34 N-(2,2-Diphenylethyl)-N′- (1- methyladamantyl)ethane-1,2-diamine

6.8 6 35 N-(2,2-Diphenylethyl)-N′- {1,1,3-trimethyl-6- azabicyclo-[3.2.1]octyl}ethane-1,2- diamine

38 30 36 N-{2-[N′-(2,2- Diphenylethyl)]- aminoethyl}- decahydroquinoline

28 24 37 N-(2,2-Diphenylethyl)-N′- (−)-cis-(myrtanyl)ethane- 1,2-diamine

54 38 38 N-(−)-cis-(Myrtanyl)-N′- (2,2-diphenylethyl)propyl- 1,2-diamine

39 30 40 N-(2,2-Diphenylethyl)-N′- (1R, 2R, 3R, 5S)-(−)-pheylethane-1,2-diamine

33 23 41 N-(−)-cis-(Myrtanyl)-N′- (2,3- dimethylcyclohexyl)ethane-1,2-diamine

66 62 42 N-(3,3-Diphenylpropyl)- N′-(−)-cis- myrtanylethane-1,2- diamine

11 9 43 N-(−)-cis-Myrtanyl-N′- (1S, 2S, 3S, 5R)-(+)-isopinocampheylethane- 1,2-diamine

31 27 47 N-(−)-cis-Myrtanyl-N′- (1R, 2R, 3R, 5S)-(−)-isopinocampheylethane- 1,2-diamine

42 33 51 N-(Cyclooctyl)-N′-(2,3- dimethylcyclohexyl)ethane- 1,2-diamine

5.1 2 52 N-(Cyctooctyl)-N′-(3,3- diphenylpropyl)ethane- 1,2-diamine

20 18 53 N-Cyclooctyl-N′-(1S, 2S, 3S, 5R)-(+)- isopinocampheyl-ethane-1,2-diamine

7.4 7 54 N-Cyclooctyl-N′-(R)-(+)- bornylethane-1,2- diamine

17 16 55 N-(Cyclooctyl)-N′-(1- methyladamantyl)ethane- 1,2-diamine

7 6 56 N-(Cyclooctyl)-N′-(2S)- [2-(1- hydroxybutyl)]ethane- 1,2-diamine

1.1 1 57 N-(−)-cis-Myrtanyl-N′- (cyclooctyl)ethane-1,2- diamine

18 18 58 N-(Cyclooctyl)-N′-(2- adamantyl)ethane-1,2- diamine

25 23 59 N-(Cyclooctyl)-N′-(1R, 2R, 3R, 5S)-(−)- isopinocampheylethane-1,2-diamine

15 14 61 N-(Cyclooctyl)-N′-[1- ethyl-(1- naphthyl)]ethane-1,2- diamine

16 14 62 N-(−)-cis-Myrtanyl-N′- (1S)-(1- ethylcyclohexane)ethane-1,2-diamine

48 46 63 N-(Cyclooctyl)-N′-trans- (2- phenylcyclopropyl)ethane-1,2-diamine

47 46 64 N-(2-Adamantyl)-N′- trans-(2- phenylcyclopropyl)ethane-1,2-diamine

49 46 65 N-(1-Adamantyl)-N′- trans-(2- phenylcyclopropyl)ethane-1,2-diamine

18 16 66 N-(3,3-Diphenylpropyl)- N′-(1R, 2R, 3R, 5S)-(−)-isopinocampheylethane- 1,2-diamine

2.3 2 68 N-(+/−)-[2-(1- Hydroxybutyl)-N′-(1R, 2R, 3R, 5S)-(−)-isopinocampheylethane- 1,2-diamine

0.8 1 71 N-(1,1-Diphenylmethyl)- N′-(1R, 2R, 3R, 5S)-(−)-isopinocampheylethane- 1,2-diamine

2.9 2 73 N-(2-Adamantyl)-N′-[2- (2- methoxyphenyl)ethyl]ethane-1,2-diamine

21 19 76 N-Allyl-N-cyclopentyl-N′- [2-(2- methoxyphenyl)ethyl]ethane-1,2-diamine

8 7 77 N-(1,1-Diphenylmethyl)- N′-[2-(2-methoxyphenyl)-ethyl]ethane-1,2-diamine

32 27 78 N-2-Adamantyl-N′-2,3- dihydro-1H-inden-2-yl- ethane-1,2-diamine

4.3 3 79 N-[2-(2,5- Dimethoxyphenyl)-ethyl]- N′-(R)-(+)-bornylethane-1,2-diamine

59 49 103 N,N′- Bis(cyclooctyl)ethane- 1,2-diamine

6.3 4 107 N-(2,2-Diphenylethyl)-N- (3-ethoxypropyl)ethane- 1,2-diamine

58 52 109 N-Geranyl-N′-(2- adamanthyl)ethane-1,2- diamine

27 24 111 N-[2-(N′- Geranyl)aminoethyl]-2- ethylpiperidine

24 24 116 N-Geranyl-N′-allyl-N′- (cyclopentyl)ethane-1,2- diamine

45 42 117 N-Geranyl-N′-(1,1- diphenyl-methyl)ethane- 1,2-diamine

24 20 118 N-2-(2- Chlorophenyl)ethyl-N′- allyl-N′-(cyclopentyl)ethane-1,2- diamine

6.4 6 119 N-2-(2- Chlorophenyl)ethyl-N′- [2-(3-fluorophenyl)-ethyl]ethane-1,2-diamine

30 27 125 N,N′-bis-(−)-cis- Myrtanylpropane-1,2- diamine

41 35 134 N-[2-(N′-2,2- Diphenylethyl)- aminoethyl]-(−)-3,4-dihydroxynorephedrine

20 15 151 N-[2-(2- Methoxy)phenylethyl]-N′- (1R, 2R, 3R, 5S)-(−)-isopinocampheyl- ethane-1,2-diamine

67 60 164 N¹-[2-(4- fluorophenyl)ethyl]-N²-[2- (4-Methoxy)phenylethyl)-1- phenylethane-1,2- diamine

94 73 165 N1-[2-(4- fluorophenyl)ethyl]-N2- (3-Phenylpropyl)-1-phenylethane-1,2- diamine

23 19

The present invention is also directed to a new library of diaminecompounds useful against infectious disease. To further enhance thestructural diversity of prior diamine compounds, a synthetic scheme toincorporate amino acids into a bridging linker between the two aminecomponents has been developed. The use of amino acids allowed fordiverse linker elements, as well as chirality see FIG. 42 forrepresentative examples. The diamine compounds were prepared on mmolscale in 96-well format in pools of 10 compounds per well (for the vastmajority of the plates). Table 25 (FIG. 43) summarizes data for thesynthesized plates.

The reaction scheme followed is shown in FIG. 44.

Solid phase syntheses using Rink resin. Twenty one 96-well plates havebeen prepared. Six-step synthetic route starting from the Rink resinsimilar to what that had been used to create our first 100,000 compoundlibrary (Scheme 1, FIG. 41), was applied to make targeted diamines(Scheme 5, FIG. 44). Overall, all steps of these schemes are similar,except one (step 4) when formation of the second amino functionalityoccurs. In Scheme 1, the second amine is introduced into the molecule asa whole synthon via nucleophilic displacement of Cl-function of thelinker, while in the Scheme 5, it proceeds through modification of theexisting amino moiety by carbonyl compounds.

Attachment of the first amine to the support was done according to theGarigipati protocol. Rink acid resin (Novabiochem) was converted intothe Rink-chloride upon treatment with triphenylphosphine anddichloroethane in THF. This activated resin was then loaded by additionof an amine N1 in presence of Hunig's base in dichloroethane. The amineN1 includes, but is not limited to, alkyl and aryl primary amines. Outof 177 primary amines that had been previously used as N1 for 100,000library preparation, only 30 were selected in this Scheme, based upon invitro activity data of their ethylenediamine derivatives (from theprevious ˜100K library) as well as structural diversity (FIGS. 45 and46).

On the next step, the acylation reaction was accomplished via peptidecoupling with FMOC protected amino acids in presence of HATU(O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluroniumhexafluorophosphate) and EtN(iso-Pr)₂ in DCM/DMF mixture at roomtemperature. The reaction was done twice to improve product yields. Thelist of the amino acids used to create this library is shown in theTable 26 (FIG. 47).

Deprotection (removal of the FMOC group) was carried out by reactionwith piperidine at room temperature. Derivatization of the amino groupwas achieved by reductive alkylation with various carbonyl compounds,such as aldehydes, ketones, and carboxylic acids, in the presence ofNaBCNH₃ at room temperature for 72-96 h. The selection of the carbonylcompounds was made so that the final diamine products would carry thesame or similar types of substituents that had been observed in the hitcompounds generated from the previous library of ethambutol analogs, aswell as structural diversity (FIG. 48). A complete list of the carbonylcompounds used is shown in Table 27 (FIG. 49).

Reduction of the aminoethyleneamides into corresponding diamines wascarried out using the soluble reducing reagent 65+w % Red-Al at roomtemperature. Cleavage of the products from the resin was achieved with a10% solution of trifluoroacetic acid in dichloromethane resulting in theformation of TFA salts of the diamines.

For library production the first three steps of the synthetic scheme(resin activation, amine loading, and acylation) were carried out usinga Quest 210 Synthesizer on scale of 0.1-0.15 g of resin per tube.Following the acylation, formed resins were thoroughly washed, dried,and then groups of ten resins were pooled together. A small amount ofeach resin (˜0.05 g) was archived prior to pooling to facilitatere-synthesis and deconvolution of actives.

Deprotection of the FMOC group, addition of the carbonyl component,reduction, and cleavage were carried out in 96-well reaction blocksusing the Combiclamps system by Whatman Polyfiltronics or the FlexChemsystem by Robbins Scientific. A suspension of the pooled resins in 2:1mixture of DCM/THF was evenly distributed into one reaction plateresulting in approximately 10 mg of the resin per well. The 96 diversecarbonyl compounds were arrayed in one 96-well plate template and added,one carbonyl compound per well, to each individual pool of ten resins,resulting in an anticipated 960 diamines produced per plate. Reductionwas carried out in the same format and cleavage and filtering intostorage plates was followed by evaporation of the TFA prior tobiological assay.

Quality assessment of the prepared compounds was done by ElectrosprayIonization mass spectrometry using two randomly selected rows (16samples) per plate, 17% of the total number. Successful production of acompound was based on an appearance of a molecular ion of the calculatedmass. Depending on the amino acid that had been used for the synthesis,the percentage of the predicted ions were observed, and therefore thepredicted compounds were formed, varied from 5-60% (Table 25, FIG. 43).Based on MS analysis, out of targeted 20,000 compounds, 4,500 diamineswere actually formed.

As discussed herein, there is a need in the art for novel compounds andmethods that are effective against infectious disease. Moreparticularly, there is a need for novel compounds and methods for theeffective treatment of Mycobacterial disease. The instant inventionsatisfies the long felt need of the prior art by providing novelcompositions and methods that are effective in the treatment ofinfectious disease, including but not limited to, tuberculosis.

In one embodiment the instant invention comprises at least two novelcompounds from Table 3 (compounds I-165) for the treatment of infectiousdisease.

In another embodiment, the instant invention comprises one or more novelcompounds of Table 3 (compounds I-165) in combination with one or moredrugs for the treatment of infectious disease.

In another embodiment, a composition comprising at least one of compoundI-165 is combined with one or more drugs for the treatment of M.tuberculosis. In a further embodiment, a composition comprising one ormore novel compounds selected from the group consisting of compoundsI-165 is combined with one or more drug to provide a synergistic effectthat is active as a method of treating Mycobacterial disease.

In one embodiment the instant invention comprises a compositioncomprising one or more compounds of Table 3 in combination with at leastone known standard tuberculosis drug.

In yet another embodiment a method of treating infectious diseasecomprises one or more compounds of Table 3 in combination with at leastone known standard tuberculosis drug. While not wishing to be bound bythe following theory it is believed that the combination of a standardtuberculosis drug with at least one or more of compounds comprisingcompounds I-165 produces a synergistic effect resulting in the treatmentor prevention of infectious disease, including but not limited to,tuberculosis.

The bactericidal activity of streptomycin, isoniazid, rifampin,ethambutol, and pyrazinamide alone and in combination againstMycobacterium Tuberculosis is discussed by Dickinson et al. (Am RevRespir Dis 116(4): 627-35): Log-phase cultures of Mycobacteriumtuberculosis in Tween-albumin medium were exposed to streptomycin,isoniazid, rifampin, ethambutol, and pyrazinamide in concentrations inthe range likely to be present in serum during treatment of patients.The bactericidal activity of the drugs was measured as the decrease inviable counts at 4 and 7 days. The activity of single drugs was highestfor streptomycin and next highest for rifampin and isoniazid, butethambutol only started to kill after 4 days. When exposed to 2 drugs,bactericidal synergism was found with streptomycin/isoniazid andisoniazid/ethambutol; additivity, with streptomycin/rifampin;indifference, with isoniazid rifampin and streptomycin/ethambutol; andantagonism, with rifampin/ethambutol and isoniazid/pyrazinamide. Whencultures were exposed to the 3 drugs, isoniazid, rifampin, andethambutol, marked antagonism was found between isoniazid and rifampin,whereas the addition of isoniazid or an increase in its concentrationincreased the bactericidal activity. Combination therapy including thenovel ethylene diamine compositions as described herein have not beenidentified prior to the present invention. The present inventioncontemplates combination therapy comprising novel ethylene diaminecompositions as presently described together with one or moreantitubercular drugs, including but not limited to rifampicin,isoniazid, pyrazinamide, moxifloxacin and ethambutol. Also includedherein are analogues and chemical equivalents and substitutes of suchantitubercular agents. For example, the present inventors contemplatethe use of rifampicin as well as its analogues, including but notlimited to, rifapentine, rifalazil and rifabutin.

In another embodiment, the present invention comprises a compositioneffective against Mycobacterium-fortuitum, Mycobacterium marinum,Helicobacter pylori, Streptococcus pneumoniae and Candida albicanscomprising at least one compound selected from the group consisting ofcompounds 1-165.

In another embodiment, the present invention comprises a compositioneffective against Mycobacterium-fortuitum, Mycobacterium marinum,Helicobacter pylori, Streptococcus pneumoniae and Candida albicanscomprising at least one compound selected from the group consisting ofcompounds I-165, alternatively combined with one or more antitubercularagents wherein the antiburcular agents, include but are not limited torifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol andanalogues thereof.

Formulations

Therapeutics, including compositions containing the substituted ethylenediamine compounds of the present invention, can be prepared inphysiologically acceptable formulations, such as in pharmaceuticallyacceptable carriers, using known techniques. For example, a substitutedethylene diamine compound is combined with a pharmaceutically acceptableexcipient to form a therapeutic composition.

The compositions of the present invention may be administered in theform of a solid, liquid or aerosol. Examples of solid compositionsinclude pills, creams, soaps and implantable dosage units. Pills may beadministered orally. Therapeutic creams and anti-mycobacteria soaps maybe administered topically. Implantable dosage units may be administeredlocally, for example, in the lungs, or may be implanted for systematicrelease of the therapeutic composition, for example, subcutaneously.Examples of liquid compositions include formulations adapted forinjection intramuscularly, subcutaneously, intravenously,intraarterially, and formulations for topical and intraocularadministration. Examples of aerosol formulations include inhalerformulations for administration to the lungs.

A sustained release matrix, as used herein, is a matrix made ofmaterials, usually polymers, which are degradable by enzymatic oracid/base hydrolysis, or by dissolution. Once inserted into the body,the matrix is acted upon by enzymes and body fluids. The sustainedrelease matrix is chosen desirably from biocompatible materials,including, but not limited to, liposomes, polylactides, polyglycolide(polymer of glycolic acid), polylactide co-glycolide (copolymers oflactic acid and glycolic acid), polyanhydrides, poly(ortho)esters,polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylicacids, fatty acids, phospholipds, polysaccharides, nucleic acids,polyamino acids, amino acids such as phenylalanine, tyrosine,isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidoneand silicone. A preferred biodegradable matrix is a matrix of one ofeither polylactide, polyglycolide, or polylactide co-glycolide.

The dosage of the composition will depend on the condition beingtreated, the particular composition used, and other clinical factors,such as weight and condition of the patient, and the route ofadministration. A suitable dosage may range from 100 to 0.1 mg/kg. Amore preferred dosage may range from 50 to 0.2 mg/kg. A more preferreddosage may range from 25 to 0.5 mg/kg. Tablets or other forms of mediamay contain from 1 to 1000 mg of the substituted ethylene diamine.Dosage ranges and schedules of administration similar to ethambutol orother anti-tuberculosis drugs may be used.

The composition may be administered in combination with othercompositions and procedures for the treatment of other disordersoccurring in combination with mycobacterial disease. For example,tuberculosis frequently occurs as a secondary complication associatedwith acquired immunodeficiency syndrome (AIDS). Patients undergoing AIDStreatment, which includes procedures such as surgery, radiation orchemotherapy, may benefit from the therapeutic methods and compositionsdescribed herein.

The following specific examples will illustrate the invention as itapplies to the particular synthesis of the substituted ethylene diaminecompounds, and the in vitro and in vivo suppression of the growth ofcolonies of M. tuberculosis. In addition, the teachings of R. Lee et al.J. Comb. Chem. 2003, 5, 172-187 are hereby incorporated by reference intheir entirety. It will be appreciated that other examples, includingminor variations in chemical procedures, will be apparent to thoseskilled in the art, and that the invention is not limited to thesespecific illustrated examples.

Example I Generating the Ethylene Diamine Library

The Rink-acid resin was obtained from NOVABIOCHEM® Inc., San Diego,Calif. Solvents: acetonitrile, dichloromethane, dimethylformamide,ethylenedichloride, methanol and tetrahydrofuran were purchased fromALDRICH®, Milwaukee, Wis., and used as received. All other reagents werepurchased from SIGMA-ALDRICH®, West Monroe Highland, Ill. Solid phasesyntheses were performed on a QUEST® 210 Synthesizer, from ARGONAUTTECHNOLOGIES®, Foster City, Calif., with the aid of combinatorialchemistry equipment, from WHATMAN® POLYFILTRONICS® (Kent, England;Rockland, Mass.) and ROBBINS SCIENTIFIC®, Sunnyvale, Calif. Evaporationof solvents was done using SPEEDVAC® AES, from SAVANT®, Holbrook, N.Y.All necessary chromatographic separations were performed on a WATERS'ALLIANCE HT SYSTEM®, Milford, Mass. Analytical thin-layer chromatographywas performed on MERCK® silica gel 60F₂₅₄ plates, purchased fromSIGMA-ALDRICH®, West Monroe Highland, Ill.

The activation of the Rink-acid resin, the addition of the first amine,and the acylation step were carried out in 10 ml tubes using the QUEST®210 Synthesizer. The addition of the second amine, the reduction withRed-AL, and the cleavage from the solid support were carried out in96-deep (2 ml) well, chemically resistant plates.

A. Activation of the Rink-Acid Resin

The Rink-acid resin had a coverage of 0.43-0.63 mmol of linker per gramresin. Four to five grams of this resin were suspended in 80 ml of a 2:1mixture of dichloromethane and tetrahydrofuran (THF), and distributedinto ten, 10 ml tubes, with 8 ml of resin suspension per tube. Eachsuspension was filtered and washed twice with THF. A solution oftriphenylphosphine (3.80 g, 14.5 mmol) in 30 ml of THF was prepared, and3 ml of this solution was added to each tube, followed by the additionof 3 ml of a solution of hexachloroethane in THF (3.39 g/14.3 mmolhexachloroethane in 30 ml THF). After agitation for six hours at roomtemperature, each activated resin was washed twice with THF and twicewith dichloromethane.

B. Addition of the First Amine

Each tube, containing the activated rink resin, was charged with 3 ml ofdichloroethane, 0.3 ml (1.74 mmol) N₁N-diisopropylethylamine (EtN(iPr)₂)and the corresponding amine (around 1 mmol). If the selected amine was asolid at room temperature, it was added as a solution, or a suspensionin DMF. Enough dichloroethane was added to each tube for a final volumeof 8 ml. The reaction mixture was heated at 45° C. for 6-8 hours. Theresins were filtered, washed with a 2:1 mixture of dichloromethane andmethanol (1×8 ml), then with methanol (2×8 ml), and then dried underargon for 10 minutes.

C. Acylation with the Halo-Acylchloride

a. Acylation with Chloroacetyl Chloride. Each resin was prewashed withTHF (2×8 ml), and then charged with THF (8 ml), pyridine (0.3 ml, 3.67mmole) and chloroacetyl chloride (0.25 ml, 2.5 mmole). The reactionmixture was stirred for 8 hours at 45° C., and then for 6-8 hours atroom temperature. Each resin was filtered, washed with a 2:1 mixture ofdichloromethane/methanol (1×8 ml), methanol (2×8 ml) and THF (2×8 ml).The acylation was repeated using the same loading of reagents, but ashorter reaction time of 4 hours at 45° C., and 2 hours at roomtemperature. Each resin was then filtered, washed with a 2:1 mixture ofdichloromethane and methanol (1×8 ml), and then with methanol (3×8 ml).Each resin was dried under argon for 10 minutes. Each resin was thentransferred into a vial and dried in a desiccator under vacuum for 1hour.

b. Acylation with α-Phenyl-α-Chloroaceryl Chloride. The same procedureset out for the acylation with chloroacetyl chloride was used. A 2.5mmol excess of α-phenyl-α-chloroacetyl chloride, relative to mmol amountof linker in the rink-acid resin, was used.

C. Acylation with α-Halo-α-Methyl; α-Halo-α-Ethyl andα-Halo-α-Butylacetyl Bromide. A 1:1:1 mixture (by volume) of theα-bromoproponyl bromide (R₄=Me), α-bromobutyryl bromide (R₄=Et), andα-bromohexanoyl bromide (R₄=Bu) was used to give a molar ratio of0.52:0.56:0.42 (in mmols). This resulted in a molar excess of 1.65, 1.75and 1.31, respectively, if the original coverage of the resin was 0.63mmol/g (0.5 g resin per tube), and 2.4, 2.6 and 1.9 if the originalcoverage of the resins was 0.43 mmol/g (0.5 g resin per tube).

d. Acylation with α-Chloro-α-Methyl Acetic acid. Each resin wasprewashed with dichloromethane. Each tube was charged with 3 ml of asolution of PyBrop (0.29 g, 0.62 mmole) in dichloromethane, a solutionof the α-chloro-α-methylacetic acid (0.095 g, 0.62 mmole) in 3 ml ofDMF, and EtN(iPr)₂ (0.2 ml, 1.2 mmole). Each reaction mixture wasallowed to react for 16-18 hours at room temperature. Each resin wasthen filtered, washed with dichloromethane (2×8 ml) and methanol (2×8ml), and the acylation was repeated. Each resin was then filtered,washed with dichloromethane (2×8 ml), methanol (3×8 ml), and dried underargon for about 10 minutes. Each resin was transferred into a vial, anddried in a desiccator under vacuum for one hour.

D. Addition of the Second Amine

Ten, or thirty prepared α-haloacetyl amide resins from the first threesteps were pooled together, leaving 0.05-0.10 gram of each individualresin for necessary deconvolutions. A suspension of the pooled resinmixture in 100 ml of a 2:1 mixture of dichloromethane and THF wasdistributed into one, two or three, 96-well reaction plates. For onereaction plate, 1.7 to 2.0 grams of resin were used. For two reactionplates, 3.0 to 3.3 grams of resin were used, and for three reactionplates, 4.7 to 5.0 grams of resin were used. The distributed suspensionwas then filtered using a filtration manifold, a small lightweightmanifold that is generally used for drawing solvents and reagents fromthe chambers of the 96-well reaction plates. The reaction plates weretransferred into COMBICLAMPS® (Huntington, W. Va.), and 10% EtN(iPr)₂ inDMF was added at 0.2 ml per well (0.21 mmole of EtN(iPr)₂ per well),followed by the addition of a 1.0M solution of the appropriate aminefrom the corresponding master plate, 0.1 ml per well (0.1 mmole amineper well). The COMBICLAMPS® are used to accommodate 96-well reactionplates during synthesis, allowing for the addition of reagents into theplates, and a proper sealing that maintains reagents and solvents forhours at elevated temperatures. These clamps consist of a top and bottomcover provided with changeable, chemically resistant sealing gaskets.They are designed to accommodate 96-well reaction plates between the topand bottom covers. The reaction plates were sealed and kept in an ovenat 70-75° C. for 16 hours. After cooling to room temperature, the resinswere filtered, washed with a 1:1 mixture of DCM/methanol (1×1 ml),methanol (2×1 ml), and then dried in a desiccator under vacuum for 2hours.

E. Reduction with Red-Al

The reaction plates were placed into COMBICLAMPS®. A 1:6 mixture ofRed-Al (65+w % in toluene) and THF was added, at 0.6 ml per well (0.28mmole of Red-Al per well), and allowed to react for 4 hours. Each resinwas then filtered, washed with THF (2×1 ml), and methanol (3×1 ml). Theaddition of methanol should proceed with caution. Each resin was thendried under vacuum.

F. Cleavage of Final Ethylene Diamine Compound

This step was carried out using a cleavage manifold, a Teflon coatedaluminum, filter/collection vacuum manifold, designed for recoveringcleavage products from the reaction plates into collection plates. Themanifold is designed to ensure that the filtrate from each well isdirected to a corresponding well in a receiving 96-well collectionplate. The reaction plates (placed on the top of the collection platesin this manifold) were charged with a 10:85:5 mixture of TFA,dichloromethane, and methanol (0.5 ml of mixture per well). Afterfifteen minutes, the solutions were filtered and collected into properwells on the collection plates. The procedure was repeated. Solventswere evaporated on a SPEED VAC®, Holbrook, N.Y., and the residualsamples (TFA salts) were tested without further purification.

Example II Deconvolution Example

Deconvolution of the active wells was performed by re-synthesis ofdiscrete compounds, from the archived α-haloacetyl amide resins (10resins, 0.05-0.10 g each), which were set aside at the end of theacylation step before the pooling. Each resin was assigned a discretecolumn (1, or 2, or 3, etc., see the template) in a 96 well filterplate,and was divided between X rows (A, B, C, etc), where X is the number ofhits discovered in the original screening plate. To each well, in a row,a selected N2 (R₃R₂NH) hit amine (0.1 mmol), DMF (180 ml) and EtNiPr₂(20 ml) were added: the first selected amine was added to the resins inthe row “A”, the second amine—to the resins in the row “B”, the thirdamine—to the resins in the row “C”, etc. A lay-out of a representative96-well filter plate is shown in Table 4.

Deconvolution of the active wells was performed by re-synthesis ofdiscrete compounds, from the archived α-haloacetyl amide resins (10resins, 0.05-0.10 g each), which were set aside at the end of theacylation step before the pooling. Each resin was assigned a discretecolumn (1, or 2, or 3, etc., see the template) in a 96 well filterplate,and was divided between X rows (A, B, C, etc), where X is the number ofhits discovered in the original screening plate. To each well, in a row,a selected N2 (R₃R₂NH) hit amine (0.1 mmol), DMF (180 ml) and EtNiPr₂(20 ml) were added: the first selected amine was added to the resins inthe row “A”, the second amine—to the resins in the row “B”, the thirdamine—to the resins in the row “C”, etc. A lay-out of a representative96-well filter plate is shown in Table 4.

The reaction plates were sealed and kept in an oven at 70-75° C. for 16hours. After cooling to room temperature, the resins were filtered,washed with a 1:1 mixture of DCM and methanol (1×1 ml), methanol (2×1ml), and dried in desiccator under vacuum for 2 h. Reduction andcleavage were performed according to steps 5 and 6 in the originalsynthetic protocol. The product wells from the cleavage were analyzed byESI-MS (Electro Spray Ionization Mass Spectroscopy) to ensure theidentity of the actives, and were tested in the same Luc and MIC assays.

TABLE 4 Lay-Out of Representative 96-Well Filter Plate A1 A2 A3 A4 A5 A6A7 A8 A9 A10 Selected amine N2, Added to A1-A10 B1 B2 B3 B4 B5 B6 B7 B8B9 B10 Selected amine N2, Added to B1-B10 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10Selected amine N2, Added to C1-C10 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10Selected amine N2, Added to D1-D10 E1 E2 E3 E4 E5 E6 E7 E8 E9 E10Selected amine N2, Added to E1-E10 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10Selected amine N2, Added to F1-F10 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10Selected amine N2, Added to G1-G10 H1 H2 H3 H4 H5 H6 H7 H8 H9 H10Selected amine N2, Added to H1-H10 *X* selected Amines N2 to be added onthe step 4 Resin Resin Resin Resin Resin Resin Resin Resin Resin ResinIndividual #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 resins ##1-10, preloaded withproper amine N1.

Example III Solid-Phase Synthesis of Selected SubstitutedEthylenediamine Compounds Using the QUEST® 210 Synthesizer

The solid-phase protocol described above in Example I was applied to thescaled-up synthesis of the selected substituted ethylene diaminecompounds. Here, all reaction steps, from the activation of theRink-acid resin to the cleavage of the final product, were carried outusing the QUEST® instrument only, which allowed for the simultaneoussyntheses of twenty parallel reactions. Purification of all crudesamples was done by HPLC to yield desirable products in purity greaterthan 90%. Table 3 lists the scale-ups of substituted ethylene diamines.Here, the synthesis of one of the active compounds,N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine is described below as anexample.

The Preparation of N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine(compound 109) is set forth in FIG. 12.

1. Activation of the Rink-acid resin. Synthesis of Rink-Cl resin.Rink-acid resin, coverage (linker) of 0.43 to 0.63 mmol/g (0.8 g, 0.5mmol), was placed into one of the 10 ml tubes of QUEST® 210 Synthesizer,and washed twice with THF. A solution of triphenylphosphine (0.380 g,1.45 mmol) in THF (3 ml) was added, followed by the addition of asolution of hexachloroethane (0.4 g, 1.43 mmol) in THF (3 ml). THF wasadded up to the volume of the tube (approximately 2 ml). After 6 hours,the resin was filtered, washed with THF (2×8 ml) and dichloromethane(2×8 ml).2. Addition of the first amine. Synthesis of resin attachedgeranylamine. The tube with activated resin was charged with 3 ml ofdichloroethane, EtN(iPr)₂, (0.3 ml, 1.74 mmol), and geranylamine (0.230g, 1.5 mmol). Dichloroethane was added to a volume of 8 ml. The reactionwas carried for 8 hours at 45° C., and for 6-8 hours at roomtemperature. Geranylamine loaded resin was filtered, washed with a 2:1mixture of dichloromethane and methanol (1×8 ml), then with methanol(2×8 ml), and suck dried for 10 minutes under argon.3. Acylation with chloroacetyl chloride. Synthesis of resin attachedN-Geranyl-α-chloroacetamide. The resin was prewashed with THF (2×8 ml).The tube was charged with 8 ml of THF, pyridine (0.3 ml, 3.67 mmol), andchloroacetyl chloride (0.2 ml, 2.5 mmol), and allowed to stir for 8 h at45° C., and 6-8 h at room temperature (RT). After the reaction wascomplete, the resin was filtered, washed with a 2:1 mixture ofdichloromethane and methanol (1×8 ml), methanol (2×8 ml), and THF, andthe acylation was repeated using the same loads of the reagents, butshorter reaction time: 4 hours at 45° C. and 2 hours at roomtemperature. At the end, the α-chloroacetamide loaded resin wasfiltered, washed with a 2:1 mixture of dichloromethane and methanol (1×8ml), methanol (3×8 ml), and suck dried for 15 min under argon.4. Addition of the second amine. Synthesis of resin attachedN-Geranyl-N′-(2-adamantyl)acetamide. The tube with the resin was chargedwith DMF (3 ml) and EtN(iPr)₂ (0.6 ml, 4.4 mmol), followed by theaddition of a suspension of 2-adamantamine hydrochloride (2.0 g, 1.1mmol) in DMF (4 ml), and was allowed to stir at 70-75° C. for 16 hours.After cooling down to the room temperature, the resin was filtered,washed with a 1:1 mixture of DCM and methanol (1×8 ml), methanol (2×8ml), and suck dried for 15 minutes under argon.5. Reduction with Red-Al. Synthesis of resin attachedN-Geranyl-N′-(2-adamantyl)ethane-1,2-diamine. The resultant resin wassuspended in anhydrous THF (3 ml) in a tube, and stirred for 15 min.Commercially available Red-Al, 65+w % in toluene, was added (2.0 ml, 6.4mmol), followed by addition of 2-3 ml of anhydrous THF (to fill up thevolume of the tube). The mixture was allowed to react for 4 hours. Afterthe reaction, the resin was filtered, washed with THF (1×8 ml), a 1:1mixture of THF and methanol (1×8 ml) (addition of MeOH should proceedwith caution), methanol (3×8 ml), and then dried.6. Cleavage from the resin and purification. Synthesis ofN-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine acetate. For this laststep of the synthesis, the tube with the resin was charged with a 10:90mixture of TFA and dichloromethane, and the formed bright red suspensionwas allowed to stir for 30 min. After addition of MeOH (0.5 ml), thecolorless suspension was filtered, and the filtrate was collected into aproper tube. The procedure was repeated, and solvents were evaporated ona SPEEDVAC®. Half of the amount of crudeN-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine (in a form oftrifluoroacetate salt) was purified by HPLC using following conditions:column C18, flow 4 ml/min, 30 min run, gradient starting with 5%AcOH/MeOH (100%) finishing up with acetonitrile (100%). Obtained: 27 mgof N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine diacetate, 24% yield,98% purity by NMR.

Example IV

Representative Solution Phase Synthesis of the Active CompoundsPreparation ofN-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine ashydrochloride (compound 59) is set forth in FIG. 13.

Bromocyclooctylacetylamide. To a mixture of cyclooctylamine (3.3 g,0.026 mol) and pyridine (2.42 g, 0.031 mmol) in anhydrous THF (80 ml) at0° C. was added dropwise, via syringe, bromoacetylbromide (5.78 g, 0.029mol). The reaction temperature was maintained by an ice bath. Thereaction mixture was allowed gradually to warm up to room temperature,and was stirred at room temperature for 1 hour. The precipitate wasremoved by filtration, washed with ethyl ether (1×30 ml), and thefiltrate was concentrated to dryness on a rotory evaporator.Bromocyclooctylacetylamide was forwarded to the second step withoutadditional purification.

N-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheyl-1-carbonylethane-1,2-diamine.To a solution of the bromocyclooctylacetylamide in DMF (60 ml) wereadded Hunig's base (4.64 g, 0.036 mol) and(1R,2R,3R,5S)-(−)-isopinocampheylamine (4.5 g, 0.029 mol), and thereaction mixture was stirred at 80° C. for 16 hours. After cooling offto the room temperature, the reaction mixture was diluted with 150 ml ofethyl ether, and washed with 1M NaOH solution (2×50 ml). The organiclayer was washed with brine (1×50 ml), dried over MgSO₄, andconcentrated to dryness on the rotory evaporator. The residue (11.04 g)as brown oil was purified on COMBIFLASK® (Isco, Lincoln, Nebr., USA),using Silicagel cartridges commercially available from BIOTAGE®(Biotage, Inc. of Dyax Corp, Va., USA), and the following mobile phasegradient: 30 min run, starting with DCM, 100%, and finishing up with amixture DCM:MeOH:N₄OH (600:400:10). The final product (7.29 g) wasobtained as a brown oil; 76% yield, purity 90%.

N-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine.To a solution of the amide, from previous step, in anhydrous THF (160ml), was added dropwise via syringe commercially available(SIGMA-ALDRICH®) Red-Al, as 65 wt % solution in THF (28 ml, 0.09 mol).The reaction mixture was stirred at reflux for 20 hours. After coolingdown to the room temperature, the reaction mixture was poured into 1.5MNaOH (200 ml), and extracted with ethyl ether (2×100 ml). The organiclayer was washed with brine (1×100 ml), dried over MgSO₄, and evaporatedto dryness on the rotory evaporator to yield 7.2 g of a crude product,as a brown oil. Chromatographic purification of the crude using the sameequipment and conditions as for the previous step, gave 3.5 g of thediamine. The diamine was treated with 2.0M solution of HCl in ethylether (25 ml), and kept in a refrigerator overnight. A dark yellow solid(4.2 g) formed, and was filtered off, and recrystallized from MeOH andethyl ether to yield 1.5 g of the diamine as an HCl salt (of puritygreater than 98%, NMR and MS are available), 19% overall yield.

Example V Mass Spectroscopy Analysis

Mass spectra data were obtained by Elecrospray Ionization technique on aPERKIN ELMER®/SCIEX®, API-300, TQMS with an autosampler, manufactured bySCIEX®, Toronto, Canada.

A. Library of Substituted Ethylenediamines

Mass spectroscopy served as a means for monitoring the reaction resultsof the library of ethylenediamines. Mass spectroscopy was done on tworandomly selected rows (24 samples) per reaction plate, for roughly28,000 compounds in pool of 10 or 30 compounds per well. Thus, if tencompounds per well were synthesized, the mass spectra for each wellshould contain ten signals, correlating with the proper molecular ionsfor each compound. The presence or absence of a particular signalindicated the feasibility of the particular synthesis. Based on the massspectral data, and on a general analysis of the reactivity of thevarious amines, it is estimated that 67,000 compounds were formed out of112,000 compounds.

FIG. 14 is a representative mass spec profile for one sample well. Massspectra for a representative ethylene diamine compound is shown in FIG.15. Tables 5 to 8, below, list illustrative examples of mass spec datafor representative reaction wells, with each well containing tensubstituted ethylene diamines.

TABLE 5 ILLUSTRATIVE EXAMPLES OF MASS SPEC DATA FOR REPRESENTATIVEETHYLENEDIAMINES (TEN COMPOUNDS PER WELL). R₂R₃NH in the 2^(nd) [M + 1]⁺of the R₁NH₂ in the 1^(st) position (pool of position (from the masterproduct 10 resins) plate of the amines) R₁NHCH₂CH₂NR₂R₃ Plate # 4-034-2,well D10 1-(2-Aminoethyl)piperidine 2-Aminoheptane 270 absentPhenethylamine 263 4-(2-Aminoethyl)morpholine 272 absent Tryptamine 302Cyclohexylamine 241 Exo-2-Aminonorbomane 253 Benzylamine 2492-Fluorophenethylamine 281 ?-Methylphenethylamine 2774-Methoxyphenethylamine 293 Plate # 4-56-1, well C4 4-Methylbenzylamineexo-2-Aminonorbornane 259 Cyclopentylamine 223 2-(Aminomethyl)piperidine246 low intensity Furfurylamine 235 3,4,5-Trimethoxybenzylamine 3351-Methyl-3-phenylpropylamine 287 Cylcobutylamine 2091,2,3,4-Tetrahydro-1- 258 naphthylamine 265 2,3-Dimethylcyclohexylamine227 low intensity 2-Amino-1-butanol Plate # 4-44-2, well G1Veratrylamine 4-Fluorophenethylamine 333 2-(1-Cyclohexenyl)ethylamine291 5-Aminoquinolone 310 absent 1-(1-Naphthyl)ethylamine 337 absent1-Aminopiperidine 266 3-Fluorobenzylamine 291 2,4-Dimethoxybenzylamine333 3-Amino-1,2,4-triazine 262 absent 2-Ethoxybenzylamine 3174-(3-Aminopropyl)morpholine 310 absent

TABLE 6 Mass Spec Data for Synthesized Ethylenediamines

[M + 1]⁺ of the products, [M + 1]⁺ of the products, R₄ = Ph R₁NH₂ in the1^(st) position R₄ = H Diamines, 1 Amino alcohols, 13 Tyramine 308 384258 formed 2-Adamantamine 321 absent 398 absent 272 formedcis-Myrtanylamine 324 400 274 formed 3-Amino-1-propanol 246 322 196absent L-Methioninol 305 absent 382 absent 256 absent Cyclooctylamine298 374 248 formed (1S,2S)-2-Amino-1- 337 absent 414 absent 288 absentphenyl-1,3-propandiol 1-Adamantane- 336 412 absent 286 formedmethylamine 368 444 318 formed 2,2-Diphenylethylamine 274 350 224 formed5-Amino-1-pentanol

TABLE 7 Mass Spec Data for Synthesized Ethylenediamines, R₄ = H and Me

[M + 1]⁺ of the products, [M + 1]⁺ of the products, R₄ = Ph R₁NH₂ in the1^(st) position R₄ = H Diamines, 1 Amino alcohols, 13 Tyramine 278 293196 absent 2-Adamantamine 293 absent 307 absent 210 lowcis-Myrtanylamine 293 intensity 3-Amino-1-propanol 217 309 212 formedL-Methioninol 277 absent 231 134 absent Cyclooctylamine 269 291 absent194 formed (1S,2S)-2-Amino-1- 309 low 269 absent 186 absentphenyl-1,3-propandiol intensity 323 absent 226 formed 1-Adamantane- 307methylamine 339 321 224 formed 2,2-Diphenylethylamine 245 353 256 formed5-Amino-1-pentanol 259 162 absent

TABLE 8 Mass Spec Data for Synthesized Ethylenediamines, R₄ = H and Me

[M + 1]⁺ of the products, [M + 1]⁺ of the products, R₄ = Ph R₁NH₂ in the1^(st) position R₄ = H Diamines, 1 Amino alcohols, 13 Tyramine 278 292absent 196 absent 2-Adamantamine 292 absent 306 absent 210 formedcis-Myrtanylamine 294 308 absent 212 formed 3-Amino-1-propanol 216 230absent 134 absent L-Methioninol 276 absent 290 absent 194 absentCyclooctylamine 268 282 absent 186 absent (1S,2S)-2-Amino-1- 308 322absent 226 formed phenyl-1,3-propandiol 1-Adamantane- 306 absent 320absent 224 formed methylamine 338 352 absent 256 formed2,2-Diphenylethylamine 244 258 absent 162 absent 5-Amino-1-pentanol

Example VI ¹H NMR Spectroscopy

Proton NMR data was recorded on a VARIAN® Nuclear Magnetic ResonanceSpectrometer (Palto Alto, Calif.) at 500 MHz.

Representative substituted ethylene diamines were purified by HPLC, andanalyzed by proton NMR. A representative proton NMR profiles is shown inFIG. 16.

NMR and MS data for some representative hit compounds are shown below.

Compound 6.

N²-(1-Adamantylmethyl)-N¹-(3,3-diphenylpropyl)propane-1,2-diamine. 55mg, 36% yield. ¹H NMR: δ 7.28-7.15 (m, 5H), 3.95 (t, J=7.9 Hz, 1H), 2.94(br s 4H), 2.71 (dd, J=7.6, 9.8 Hz, 2H), 2.41 (s, 2H), 2.32 (dd, J=7.6,7.9 Hz, 2H), 2.16 (s), 2.08-1.98 (m, 4H), 1.72 (m, 6H), 1.62 (m, 6H),1.51 (d, J=2.4 Hz, 3H). Mass spectrum (ESI) m/z (MH)⁺417.

Compound 7.

N-(3,3-Diphenylpropyl)-N′-(1-adamanthylmethyl)ethane-1,2-diamine. 28 mg,22% yield. ¹H NMR (500 MHz) δ 7.30-7.12 (m, 10H); 3.95 (t, J=7.6 Hz,1H); 2.91 (d, J=1.2 Hz, 4H); 2.70 (dd, J=7.6 and 1.2 Hz, 2H); 2.40 (d,J=1.3 Hz, 2H); 2.32 (q, J=8.0 Hz, 2H); 1.98 (br d, J=1.7 Hz, 4H); 1.72(d, J=12.2 Hz, 4H); 1.62 (d, m? J=12.2 Hz, 4H); 1.51 (br s, 6H). Massspectrum (ESI) m/z (MH)⁺403.6.

Compound 10.

N-(−)-cis-Myrtanyl-N′-(3,3-diphenylpropyl)ethane-1,2-diamine. 14 mg, 11%yield. ¹H NMR (500 MHz) δ 7.30-7.10 (m, 10H); 3.95 (m, 1H); 2.92-2.83(m, 4H); AB: 2.80 (d, J=7 Hz, 1H); 2.76 (d, J=8 Hz, 1H); 2.65 (dd, J=9.6and 7.6 Hz, 2H); 2.42-2.20 (m, 4H), 2.29 (d, J=8 Hz, 2H), 1.90 (m, 8H);1.42 (m, 1H); 1.19 (m, 2H); 1.17 (s, 3H); 0.95 (s, 3H); 1.00-0.8 (m,2H). Mass spectrum (ESI) m/z (MH)⁺391.3.

Compound 14.

N-(3,3-Diphenylpropyl)-N′-exo-(2-norborny)ethane-1,2-diamine. 17 mg, 16%yield. ¹H NMR (500 MHz) δ 7.30-7.15 (m, 10H); 3.95 (t, J=7.9 Hz, 1H);2.86 (dd, J=11.5 and 1.5 Hz, 4H); 2.73 (dd, J=8.0 and 3.3 Hz, 1H); 2.64(t, J=7.6 Hz, 2H); 2.29 (t, J=7.5 Hz, 2H), 2.31-2.26 (m, 2H) 2.30 1.96(s, 3H); 1.63 (ddd, J=13.1, 7.9 and 2.5 Hz, 1H); 1.60-1.50 (m, 1H);1.50-1.43 (m, 2H); 1.30 (dq, J=4.0 and 13.5 Hz, 1H), (1H, m), 1.20 (dd,J=10.4 and 1.1 Hz, 1H), 1.11 (dd, J=2.0, and 8.5 Hz, 1H), 1.08 (dd,J=2.5, and 8.5 Hz, 1H), 1.10 (dq, J=8.3 and 2.1, 2H). Mass spectrum(ESI) m/z (MH)⁺349.1.

Compound 21.

N-(3,3-Diphenylpropyl)-N′-(1S)-(1-ethylcyclohexane)ethane-1,2-diamine. 5mg, 4% yield. Mass spectrum (ESI) m/z (MH)⁺365.5.

Compound 32.

N-(2,2-Diphenylethyl)-N′-®-(+)-bornylethane-1,2-diamine. 58 mg, 48%yield. ¹H NMR (500 MHz): δ 7.30-7.10 (m, 10H); 4.18 (t, J=6.8 Hz, 1H);3.34 (d, J=7.6 Hz, 2H); 3.02 (m, 4H); 2.95-2.90 (m, 1H); 2.15-2.08 (m,1H); 1.94 (m, 1H); 1.72-1.65 (m, 2H); 1.48-1.30 (m, 2H); 1.27-1.10 (m,2H); 1.06 (dd, J=13.6 and 4.1 Hz, 1H); 0.82 (s, 3H); 0.81 (s, 3H); 0.78(s, 3H). Mass spectrum (ESI) m/z (MH)⁺377.2

Compound 34.

N-(2,2-Diphenylethyl)-N′-(1-adamanthylmethyl)ethane-1,2-diamine. 6.8 mg,6% yield. ¹H NMR (500 MHz) δ 7.30-7.15 (m, 10H); 4.15 (t, J=7.6 Hz, 1H);3.24 (dd, J=7.9 and 1.2 Hz, 2H); 2.79 (t, J=6.5 Hz, 2H); 2.74 (t, J=6.0Hz,m, 2H); 1.95 (m, 8H); 1.69 (d, J=12.5 Hz, 4H); 1.59 (d, J=11.9 Hz,4H); 1.40 and 1.39 (br s, 3H); Mass spectrum (ESI) m/z (MH)⁺389.0.

Compound 37.

N-(2,2-Diphenylethyl)-N′-(−)-cis-myrtanylethane-1,2-diamine. 54 mg, 38%yield. ¹H NMR: δ 7.31-7.18 (m, 10H), 4.13 (t, J=7.6 Hz, 1H), 3.26 (d,J=7.6 Hz, 2H), 2.86 (dd, J=4.3, 8.0 Hz, 4H), 2.76 (dd, J=7.6, 12.2 Hz,2H), 2.37 (ddd, J=1.8, 9.0, 12.5 Hz, 1H), 2.12 (dq, J=1.8, 7.6 Hz, 1H),1.98 (br s, 2H), 1.98-1.84 (m, 4H), 1.39 (ddd, J=2.4, 4.0, 6.1 Hz, 1H),1.18 (s, 3H), 0.95 (s, 3H), 0.91 (d, J=10.0 Hz, 1H) Mass spectrum (ESI)m/z (MH)⁺377.2.

Compound 38.

N-(−)-cis-Myrtanyl-N′-(2,2-diphenylethyl)propane-1,2-diamine. 39 mg, 30%yield. 1H NMR (500 MHz) δ 7.30-7.15 (m, 10H); 4.13 (t, J=8.0 Hz, 1H);AB: 3.28 (d, J=7.5 Hz, 1H); 3.24 (d, J=7.5 Hz, 1H), 3.26 (d, J=6.1 Hz,2H); 2.96 (m, 1H); 2.88-2.75 (m, 2H); 2.71 (ddd, J=4.5, 9.0, 13.0 Hz,1H), 2.58 (ddd, J=7.0, 10.0, 14.0 Hz, 1H); 2.35 (m, 1H); 2.21 (m, 1H);2.00-1.80 (m, 6H); 1.40-1.20 (m, 1H); 1.17 (s, 3H); 0.93 (s, 3H); 0.89(dd, J=9.7 and 4.2 Hz, 1H). Mass spectrum (ESI) m/z (MH)⁺391.0.

Compound 40.

N-(2,2-Diphenylethyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine.33 mg, 23% yield. ¹H NMR: δ 7.31-7.18 (m, 10H), 4.13 (t, J=7.5 Hz, 1H),3.27 (d, J=8.0 Hz, 2H), 3.14 (dt, J=6.0, 10 Hz, 1H), (4H), 2.36 (qd,J=2.0, 6.0 Hz, 1H), 2.34 (dt, J=2.0, 10 Hz, 1H), 2.07-1.96 (m, 3H), 1.82(dt, J=2.0, 6.0 Hz, 1H), 1.71 (ddd, J=2.5, 5.5, 13.5 Hz, 1H), 1.22 (s,3H), 1.09 (d, J=7.0 Hz, 3H), 0.96 (d, J=10.5 Hz, 1H), 0.91 (s, 3H). Massspectrum (ESI) m/z (MH)⁺377.3.

Compound 47.

N-(−)-cis-Myrtanyl-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine.42 mg, 33% yield. ¹H NMR: 33.35-3.20 (m, 6H), 2.93 (dd, J=4.6, 2.0 Hz,2H), 2.45-2.33 (m, 4H), 2.17 (s, 3H), 2.06 (quint, J=7.0 Hz, 1H),2.0-1.9 (m, 6H), 1.90 (dd, J=2.1, 5.2 Hz, 1H), 1.87 (dt, J=1.8, 4.6 Hz,1H), 1.51 (ddd, J=4.6, 10.0, 13.0 Hz, 1H), 1.23 (s, 3H), 1.19 (s, 3H),1.12 (d, J=8 Hz, 3H), 1.03 (d, J=10.3 Hz, 1H), 0.98 (s, 3H), 0.94 (d,J=9.8 Hz, 1H), 0.94 (s, 3H). Mass spectrum (ESI) m/z (MH)⁺333.6.

Compound 52.

N-(3,3-Diphenylpropyl)-N′-cyclooctylethane-1,2-diamine. 20 mg, 18%yield. ¹H NMR (500 MHz): δ 7.30-7.10 (m, 10H); 3.96 (t, J=7.9 Hz, 1H);3.00 (m, 1H); 2.90 (dd, J₁=J₂=5.5 Hz, 2H); 2.84 (dd, J₁=J₂=5.0 Hz, 2H);2.61 (t, J=7.3 Hz, 2H), 2.27 (q, J=7.6 Hz, 2H); 1.83 (m, 2H); 1.74 (m,2H); 1.65-1.40 (m, 10H).

Compound 55.

N-(1-Adamantylmethyl)-N′-cyclooctylethane-1,2-diamine. 6.7 mg, 6% yield.¹H NMR (500 MHz): δ 3.08-3.02 (m, 1H), 3.02-2.98 (m, 2H); 2.97-2.92 (m,2H); 2.36 (s, 2H); 1.98 (m, 2H); 1.93-1.86 (m, 2H); 1.80-1.50 (m, 19H).

Compound 57.

N-(−)-cis-Myrtanyl-N′-(cyclooctyl)ethane-1,2-diamine. 18 mg, 18% yield.¹H NMR (500 MHz) δ 3.05-2.95 (m, 4H); AB: 2.76 (d, J=7.5 Hz, 1H), 2.23(d, J=8.0 Hz, 1H); 2.76 (dd, J=11.6 and 7.3 Hz, 1H); 2.73 (dd, J=11.9and 8.2 Hz, 1H); 2.40-2.34 (m, 1H); 2.28 (quintet, J=8.0 Hz, 1H); 1.97(s, 3H); 2.00-1.84 (m, 6H); 1.80-1.70 (m, 2H); 1.68-1.38 (m, 1H); 1.18(s, 3H); 0.97 (s, 3H); 0.92 (d, J=9.8 Hz, 1H). Mass spectrum (ESI) m/z(MH)⁺307.5.

Compound 58.

N-(2-Adamantyl)-N′-cyclooctylethane-1,2-diamine. 25 mg, 23% yield. ¹HNMR: δ 3.06 (m, 1H), 3.00 (t, J=6.1 Hz, 2H), 2.93 (t, J=5.5 Hz, 2H),2.83 (br s, 1H), 1.96 (s, 3H), 1.92-1.80 (m, 10H), 1.80-1.50 (m, 20H).Mass spectrum (ESI) m/z (MH)⁺305.1.

Compound 59.

N-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine.15 mg, 14% yield. ¹H NMR (400 MHz): δ 3.47 (dt, J=6.0, 10.0 Hz, 1H),3.40-3.28 (m, 7H), 2.44 (tq, J=2.0, 10.0 Hz, 1H), 2.36 (dtd, J=2.0, 6.0,10.0 Hz, 1H), 2.09 (dq, J=2.0, 7.2 Hz, 1H), 2.00-1.90 (m, 3H), 1.88-1.78(m, 2H), 1.78-1.63 (m, 4H), 1.65-1.30 (m, 8H), 1.18 (d, J=6.0 Hz, 3H),1.16 (s, 3H), 1.17 (d, J=7.2 Hz, 1H), 0.90 (s, 3H). Mass spectrum (ESI)m/z (MH)⁺307.4.

Compound 62.

N-(−)-cis-Myrtanyl-N′-(1S)-(1-ethylcyclohexane)ethane-1,2-diamine. 48mg, 46% yield. ¹H NMR (500 MHz): δ 3.06-3.00 (m, 1H); 2.98-2.95 (m, 2H);2.92-2.84 (m, 1H); 2.79 (dd, J=11.9 and 7.0 Hz, 1H); 2.75 (dd, J=11.9and 7.9 Hz, 1H); 2.73 (m, 1H); 2.39 (m, 1H); 2.28 (quintet, J=8.5 Hz,1H); 2.00-1.86 (m, 6H); 1.82-1.76 (m, 2H); 1.68 (m, 2H); 1.54-1.42 (m,2H); 1.32-1.10 (m, 6H); 1.19 (s, 3H); 1.13 (d, J=6.7 Hz, 3H); 1.07 (dd,J=12 and 3 Hz, 2H); 1.02 (dd, J=12 and 3 Hz, 2H); 0.98 (s, 3H); 0.93 (d,J=9.7 Hz, 1H). Mass spectrum (ESI) m/z (MH)⁺306.9.

Compound 65.

N-trans-(2-phenylcyclopropyl)-N′-(1-adamanthyl)ethane-1,2-diamine. 18mg, 16% yield. Mass spectrum (ESI) m/z (MH)⁺311.3.

Compound 66.

N-(3,3-Diphenylpropyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine.2 mg, 2% yield. ¹H NMR (500 MHz) δ 7.26 (m, 10H); 3.96 (t, J=7.6 Hz,1H); 3.09 (m, 1H); 2.92 (m, 1H); 2.84 (m, 2H); 2.62 (m, 2H); 2.35 (m,4H); 1.97 (s, 3H); 1.82 (m, 1H); 1.68 (m, 1H); 1.21 (s, 3H); 1.12 (d,J=7.3 Hz; 3H); 1.01 (m, 1H); 0.92 (s, 3H). Mass spectrum (ESI) m/z(MH)⁺391.4.

Compound 73.

N-(2-Adamantyl)-N′-[2-(2-methoxyphenyl)ethyl]ethane-1,2-diamine. 21 mg,19% yield. ¹H NMR: δ 7.22 (dd, J=8.2, 7.3 Hz, 1H), 7.14 (d, J=7.3 Hz,1H), 6.89 (d, J=7.1, Hz, 1H), 6.87 (d, J=8.2, Hz, 1H), 3.81 (s, 3H),3.06 (t, J=7.1 Hz, 2H), 3.06 (m, 2H), 3.01 (m, 2H), 2.93 (t, J=7.1, 2H),1.95 (br s, 2H), 1.90-1.80 (m, 7H), 1.78-1.66 (m, 6H), 1.59 (d, J=2.5Hz, 2H). Mass spectrum (ESI) m/z (MH)⁺329.4.

Compound 78.

N²-Adamantyl-N′-2,3-dihydro-1H-inden-2-yl-ethane-1,2-diamine. 4.3 mg, 3%yield. ¹H NMR: δ 7.20 (dd, J=4.9, 8.5 Hz, 2H), 7.14 (dd, J=5.5, 2.1 Hz,2H), 3.71 (quint, J=6.1 Hz, 2H), 3.19 (dd, J=5.8, 15.9 Hz, 2H), 3.13(br.s, 1H), 3.05 (m, 4H), 2.86 (dd, J=4.8, 15.8 Hz, 2H), 2.08 (m, 2H),2.00 (m, 6H), 1.96-1.88 (m, 4H), 1.88-1.80 (m, 3H). 1.74 (m, 4H),1.68-1.60 (m, 2H). Mass spectrum (ESI) m/z (MH)⁺303.4.

Compound 109.

N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine. 27 mg, 24% yield. ¹H NMR(400 MHz): δ 5.40 (t, J−7.2 Hz, 1H), 4.78 (br s, 2H), 3.64 (d, J=7.6 Hz,2H), 3.34 (m, 2H), 2.07 (m, 2H), 2.08-1.95 (m, 4H), 1.95-1.85 (m, 4H),1.82 (m, 2H), 1.88-1.70 (m, 4H), 1.70-1.62 (m, 3H), 1.67 (s, 3H), 1.56(s, 3H), 1.50 (s, 3H). Mass spectrum (ESI) m/z (MH)⁺307.4.

Compound 111.

N-Geranyl-N′-(2-ethylpiperidine)ethane-1,2-diamine. 44 mg, 42% yield. 1HNMR (500 MHz): δ 5.22 (t, J=6.1 Hz, 1H); 5.04 (m, 1H), 3.52 (d, J=7.3Hz, 2H); 3.05-2.85 (m, 4H); 2.66 (m, 1H); 2.44 (m, 2H); 2.08 (m, 4H);1.80-1.50 (m, 2H); 1.70 (s, 3H); 1.65 (s, 3H); 1.58 (s, 3H); 1.50-1.35(m, 2H), 0.89 (t, J=7.3, 3H). Mass spectrum (ESI) m/z (MH)⁺293.4

Compound 116.

N-Geranyl-N′-allyl-N′-(cyclopentyl)ethane-1,2-diamine. 45 mg, 42% yield.¹H NMR: δ 5.86 (ddd, J=10.0, 16.1, 6.7 Hz, 1H), 5.28 (d, J=15.9 Hz, 1H),5.25 (d, J=8.7 Hz, 1H), 5.23 (t, J=7.3 Hz, 1H), 5.30 (m, 1H), 3.59 (d,J=7.3 Hz, 2H), 3.28 (br d, J=6.4 Hz, 2H), 3.16 (quintet, J=8.2 Hz, 1H),3.02 (m, 2H), 2.95-2.86 (m, 2H), 1.88-1.80 (m, 4H), 1.70 (s, 3H),1.74-1.66 (m, 3H), 1.65 (s, 3H), 1.58 (s, 3H), 1.56-1.50 (2H), 1.50-1.40(m, 2H). Mass spectrum (ESI) m/z (MH)⁺305.3.

Compound 117.

N-Geranyl-N′-diphenylmethylethane-1,2-diamine. 24 mg, 20% yield. 1H NMR(500 MHz): δ 7.40 (d, J=7.2 Hz, 4H); 7.29 (t, J=7.3 Hz, 4H); 7.21 (t,J=7.0 Hz, 2H); 5.15 (t, J=7.5, 1H); 5.01 (m, 1H); 4.89 (br s, 1H); 3.42(d, J=7.0 Hz, 2H); 3.00-2.78 2.93 (m, 4H); 2.20-2.00 2.17 (m, 4H); 1.63(s, 3H); 1.59 (s, 3H); 1.56 (s, 3H). Mass spectrum (ESI) m/z (MH)⁺363.3.

Compound 125.

N,N′-bis-(−)-cis-Myrtanylpropane-1,2-diamine. 82 mg, 70% yield. ¹H NMR(500 MHz): δ 3.62 (m, 1H); 3.18 (dd, J=13.7 and 3.7 Hz, 1H); 3.05 (dt,J=11.5 and 7.5 Hz, 1H); 3.06-2.92 (m, 2H); 2.86 (dt, J=12.2 and 7.3 Hz,1H); 2.40 (m, 4H); 2.06-1.84 (m, 10H); 1.56-1.46 (m, 2H); 1.37 and 1.36(two d, J=6.7 and J=7.0 Hz, 3H); 1.20 (s, 3H); 1.19 (m, 3H), 0.99 and0.98 (two s, 3H) Hz, H); 0.97 (s, 3H); 0.94 (two d, J=10.1 Hz, 2H). Massspectrum (ESI) m/z (MH)⁺346.9.

Compound 151.

N-[2-(2-Methoxy)phenylethyl]-N′-(1R,2R,3R,5S)-(−)-isopinocampheyl-ethane-1,2-diamine.67 mg, 60% yield. ¹H NMR (500 MHz): δ 7.23 (t, J=5.8 Hz, 1H); 7.13 (dd,J=5.8 and 1.8 Hz, 1H); 6.88 (m, 2H); 3.81 (s, 3H); 3.13 (m, 1H); 3.1-3.0(m, 3H); 3.01 (t, J=7.0 Hz, 2H); 2.89 (t, J=7.0 Hz, 2H); 2.42-2.35 (m,2H); 2.00 (m, 3H); 1.82 (dt, J=6.0 and 2.0 Hz, 1H); 1.72 (ddd, J=2.5,5.5, 13.5 Hz, 1H); 1.22 (s, 3H) 1.13 (d, J=7.3 Hz, 3H). 0.99 (d, J=10.1Hz, 1H); 0.93 (s, 3H). Mass spectrum (ESI) m/z (MH)⁺331.5.

N²-(2-Methoxyphenyl)ethyl-N′-allyl-N′-cyclopentyl-ethane-1,2-diamine. 8mg, 7% yield. ¹H NMR: δ 7.26 (dd, J=7.3, 8.5, 1H), 7.18 (d, J=7.2 Hz,1H), 6.91 (m, 2H), 5.61 ddd, (J=6.7, 17.0, 9.4 Hz, 1H), 5.13 (d, J=15.3Hz, 1H), 5.10 (d, J=9.2 Hz, 1H), 3.83 (s, 3H), 3.13 (dd, J=7.0, 6.7 Hz,2H), 3.10 (d, J=6.7 Hz, 1H), 3.00 (d, J=7.3 Hz, 1H), 3.05-2.90 (m, 2H),2.97 (dd, J=8.2, 6.1 Hz, 2H), 2.75 (t, J=6.1 Hz, 2H), 1.73 (m, 2H), 1.62(m, 2H), 1.50 (m, 2H), 1.22 (m, 2H). Mass spectrum (ESI) m/z (MH)⁺311.4.

N²-(3-Phenylpropyl)-N′-[2-(4-fluorophenyl)ethyl]-1-phenylethane-1,2-diamine.23 mg, 19% yield. ¹H NMR: δ 7.35 (d, J=7.6 Hz, 2H), 7.34 (quart, J=7.Hz, 1H), 7.26 (d, J=6.4 Hz, 3H), 7.23 (d, J=7.6 Hz, 2H), 7.17 (dd,J=7.3, 6.4 Hz, 1H), 7.12 (d, J=7.0 Hz, 2H), 3.21 (m, 1H), 3.03 (ddd,J=4.2, 8.0, 12.8 Hz, 4H), 2.86 (t, J=8.0 Hz, 2H), 2.85-2.79 (m, J=12.Hz, 2H), 2.74-2.64 (m, 4H), 2.61 (t, J=7.7 Hz, 2H), 1.96 (quint, J=7.6Hz, 2H). Mass spectrum (ESI) m/z (MH)⁺377.3.

Example VII M. Tuberculosis Rv0341p Lucs Drug Response

Substituted ethylene diamines, as described herein, were tested onMycobacterium tuberculosis using high-throughout screening assay withrecombinant mycobacterial containing promoter fusion of luciferase toRv0341EMB-inducible promoter. This assay quickly and reliably identifiesantimycobacterial activity in compound mixtures and/or in individualcompounds. In this assay, bioluminescence increases when themycobacteria is tested against an active compound, or an active compoundmixture. During this assay, a theoretical yield of 100% was assumed forevery unpurified substituted ethylene diamine, and the activity of eachsample was compared to commercially available ethambutol (99.0% purity).Results were reported in LCPS, and % Max. LCPS based on the activity ofEMB at 3.1 μM.

The substituted ethylene diamines were analyzed according to thefollowing procedure. The diamines were dried in a speed vacuum to anapproximate concentration of 6.3 mmoles per well. Each diamine, ordiamine mixture, was then resuspended or dissolved in 200 μl of methanolfor a concentration of 31.5 mM diamine(s). The diamine(s) solution wasdiluted to a concentration of 200 μM in 7H9 broth medium (a 1:15.75dilution of the 31.5 mM stock, followed by a 1:10 dilution; eachdilution in 7H9 broth medium). Next, 50 μl of the diluted diamine(s)solution was added to the first well of a row of twelve in an opaque,96-well plate. The 7H9 broth medium, 25 μl, was added to each of theremaining wells (#2-12) in the row. The diamine(s) solution in “wellone” was serially diluted by transferring 25 μl from “well one” to “welltwo”, and repeating a 25 μl transfer from “well two” to “well three”,and so on, on through “well eleven”. In “well eleven”, the extra 25 μlof solution was discarded. “Well twelve” was used as a growth control toassess background activity of the reporter strain. The plate was thencovered and incubated at 37° C. for 24 hours. Immediately prior toanalysis, the following substrates were prepared: a buffer solutioncontaining 50 mM HEPES at pH 7.0 and 0.4% Triton X-100. Then, 0.25 ml of1M DTT, and 14 μl of 10 mg/ml luciferin in DMSO were added to 5 ml ofthe buffer solution. This final solution (50 μl) was added to each ofthe twelve wells, immediately after the incubation period had run. Theluminescence from each well was measured 20 minutes after the luciferinsubstrate was added, using a TOPCOUNT® (Downers, Grove, Ill.) NXTluminometer (55/well).

FIGS. 6-8 show typical assay data for the luciferase reporter straincontaining an Rv0341 EMB-inducible promoter with serial dilution of 12wells (1 row) of a 96-well library plate. FIG. 10 shows the number ofsubstituted ethylene diamines with at least 10% luciferase activity,based on the activity of ethambutol at 3.1 μM.

FIG. 6 represents typical assay data in the luciferase reporter straincontaining an Rv0341 EMB-inducible promoter. The data represents valuesobtained from the HTS Luc assay for compound mixtures of one row (row D)in the 96-well library. Row D was subject to several serial dilutions.The effectiveness of the compound mixture in the assay was measured bythe intensity of luminescence, and compared to ethambutol (100%intensity, 99% purity) at 3.1 μM. Each curve in FIG. 6 represents onewell, or ten compounds. Results are reported in percent maximumLuminescence Count per Second (% Max. LCPS). During the screening, atheoretical 100% chemical yield was assumed for every unpurifiedcompound. Concentrations are given for a single compound. Based on thisinitial screening, 300+ compound mixtures showed anti-TB activity

Example VIII Representative MIC Experiment

The Minimum Inhibition Concentration (MIC) is the concentration of thegrowth inhibitor, here a substituted ethylene diamine, at which there isno multiplication of seeded cells. A microdilution method was used todetermine the MIC of the substituted ethylene diamines, capable ofinhibiting the growth of Mycobacterium tuberculosis in vitro. In arepresentative MIC experiment, bacteria, the H37Rv strain ofMycobacterium tuberculosis (M.tb), was cultivated in 7H9 medium to adensity of 0.2 OD (optical density) at 600 nm. The bacterial culture wasthen diluted 1:100 in 7H9 broth medium. Stock solutions of isoniazid andethambutol were each prepared at 32 μg/ml in 7H9 medium. A 3.2 mg/mlsolution of isonizid and ethambutol were each prepared in water. Thesolutions were then filtered, and diluted 1:100 in 7H9 medium. Eachdrug, purchased from Sigma, was “laboratory use only” grade. A 10 mMsolution of each substituted ethylene diamine was prepared in methanol.Next, 100 μl of the 7H9 medium was added to each well in a 96-well plate(rows (A through H)×columns (1 through 12)). To the first wells in rowsC through H was added an additional 80 μl of the 7H9 medium. Theisoniazid solution, 100 μl, was added to well A1, and the ethambutolsolution, 100 μl, was added to well B1. Six substituted ethylenediamines, 20±1 each, were added to wells C1 through H1 (column 1),respectively. A serial dilution of each substituted ethylene diamine andthe isoniazid and ethambutol controls, was performed across each row.For example, a serial dilution across row C1-C12 was done by mixing andtransferring 100 μl of the previous well to the next consecutive well.In each well in “column 12,” 100 μl of the final dilution was discarded.Next, 100 μl of the diluted H37Rv strain of M.tb was added to each well.The 96-well plate was then covered and incubated at 37° C. for 10 days.The plate was read for bacterial growth, or non-growth, using aninverted plate reader. The MIC was determined to be the lowestconcentration of substituted ethylene diamine that inhibited visiblegrowth of the M. tuberculosis.

A representative plate layout, listing concentration in each well, isshown in Table 9. Table 10 lists MIC and LD50 data for selectedcompounds. The LD50 is the concentration of the substituted ethylenediamine at which 50% of the cells (H37Rv strain of M.tb) are killed.Table 11 lists MIC data for purified substituted ethylene diamines incomparison to ethambutol (EMB). FIG. 9 shows the number of substitutedethylene diamine compounds with MIC activity at various concentrationlevels.

TABLE 9 Concentration in Each Well (μM) Based on Columns 1-12 DRUGIsoniazid 58.25 29.13 14.56 7.28 3.64 1.82 0.91 0.45 0.23 0.11 0.06 0.03Ethambutol 28.75 14.38 7.19 3.60 1.80 0.90 0.45 0.22 0.11 0.06 0.03 0.01Subst. 500 250 125 62.5 31.25 15.63 7.81 3.91 1.96 0.98 0.49 0.24Ethylene Diamine

TABLE 10 Selectivity Index for Selected Compounds MIC LD50 LD50 Cmpd(uM) (uM) MW MIC (ug/ml) (ug/ml) SI 6 7.813 20 536 4.187768 10.722.559836 34 7.813 32 508 3.969004 16.256 4.095738 37 15.625 32 496 7.7515.872 2.048 47 15.625 25 452 7.0625 11.3 1.6 57 15.625 18 426 6.656257.668 1.152 59 15.625 32 426 6.65625 13.632 2.048 65 15.625 60 4306.71875 25.8 3.84 109 1.953 32 450 0.87885 14.4 16.38505 111 7.813 44412 3.218956 18.128 5.63164 151 7.813 41 450 3.51585 18.45 5.247664

The above procedure was also used to examine batched compounds (10compounds per well). Synthesized batches of substituted ethylenediamines were dried in speed vacuum and then resuspended in DMSO orsterile water to a concentration of 2.5 mg/ml.

TABLE 11 MIC Data for Purified Samples Plate set-up INH 58.25 29.12514.56 7.28 3.64 1.82 0.91 0.45 0.23 EMB 28.75 14.375 7.1875 3.594 1.7970.898 0.449 0.2245 0.1125 CMPD 500 250 125 62.5 31.25 15.625 7.8133.9063 1.953 Avg INH MIC (uM) Avg INH MIC (uM) 0.91  0.91 Avg EMB MIC(uM) Avg EMB MIC (uM) Avg EMB Avg EMB 7.1875 8.37 7.25 7.25 BACTEC (EMB:Cmpd MIC (uM) 2.5 UG/ML) 1 250 250 125 125 2 250 250 250 250 3 31.2562.5 15.6 15.6 4 125 62.5 62.5 62.5 5 >500 500 500 500 6 7.813* 7.8133.9 3.9 7 15.625* 7.813 3.9 3.9 8 125 125 31.25 31.25 10 7.813* 15.6257.8 7.8 11 31.25 contaminated 3.9 3.9 13 31.25 31.25 15.6 15.6 15 1415.625″ 15.625 7.8 7.8 15 >500 >500 250 500 17 62.5 62.5 15.6 15.6 2115.625* 31.25 7.8 7.8 22 31.25 31.25 7.8 15.6 23 31.25 31.25 15.6 15.624 125 125 31.25 31.25 27 125 62.5 15.6 31.25 28 125 62.5 31.25 31.25 2962.5 62.5 31.25 62.5 31 31.25 61.25 15.6 15.6 32 15.625* 15.625 7.8 7.833 62.5 62.5 31.25 31.25 34 7.813* 7.813 3.9 3.9 35 62.5 62.5 15.6 31.2536 31.25 62.5 15.6 15.6 37 15.625* 15.625 3.9 7.8 1.25 38 7.813 7.8133.9 7.8 40 15.625* 15.625 7.8 7.8 41 31.25 15.625 15.6 15.6 42 31.2531.25 1.95 3.9 43 31.25 31.25 3.9 7.8 12.5 47 15.625* 15.625 1.95 7.8 551 31.25 250 31.25 31.25 52 15.625* 15.625 3.9 3.9 53 31.25 31.25 31.2531.25 54 31.25 31.25 15.6 31.25 55 15.625* 15.625 15.6 15.6 25 56500 >500 500 500 57 15.625* 7.813 7.8 7.8 58 15.625* 15.625 7.8 7.8 5 5915.625* 31.25 15.6 15.6 12.5 61 62.5 62.5 31.25 31.25 62 15.625* 31.2515.6 31.25 63 62.5 62.5 31.25 62.5 64 31.25 31.25 31.25 31.25 65 15.625*31.25 31.25 31.25 66 15.625* 15.625 7.8 7.8 68 500 500 500 500 71 62.562.5 31.25 31.25 73 62.5 15.6 15.6 76 62.5 62.5 31.25 31.25 77 31.2531.25 15.6 15.6 78 15.625* 31.25 15.6 15.6 79 31.25 31.25 15.6 15.6 10331.25 31.25 62.5 62.5 107 500 500 250 250 109 1.953* 1.953 1.95 1.950.63 111 7.813* 7.813 7.8 7.8 5 116 15.625* 15.625 7.8 15.6 12.5 1177.813* 15.625 7.8 7.8 118 31.25 62.5 31.25 no data 119 125 contam 62.5cont no data 125 15.625* 15.625 cont no data 6.25 134 >500 >500 500 nodata 151 15.625* 7.813 cont no data 6.25 164 62.5 125 cont no data 16562.5 62.5 15.6 15.6

Example IX Secondary Screening and Evaluation of Substituted EthyleneDiamines Against Drug Resistant Patient Isolates

Secondary screening was performed on some of the substituted ethylenediamine compounds to examine their activity against three clinicallyresistant MDR patient isolates. MDR Strain TN576 is classified as a W1strain (STP^(R), INH^(R), RIF^(R), EMB^(R), ETH^(R), KAN^(R), CAP^(R))strain TN587 is classified as a W strain (STP^(R), INH^(R), RIF^(R),EMB^(R), KAN^(R)), and the third strain TN3086 is classified as a W1strain (STP^(R), INH^(R), RIF^(R), EMB^(R), KAN^(R)). Each MDR strain ishighly resistant to ethambutol with MIC values exceeding 12.5-25 μM. TheMICs for the following substituted ethylene diamines, MP 116, MP 117, RL241, compounds #59 and #109, were determined for all three patientisolates.

The results from this study are shown in Tables 12-13. Table 14characterizes each MDR strain according to its resistance.

TABLE 12 Screening of Substituted Ethylene Diamines Against DrugResistant Patient Isolates - (MIC values in ug/ml) WT 576 587 3806 EMB3.12 (or 11.1 uM) 12.5-25 12.5-25 12.5-25 MP 116 6.25 3.15 6.25 3.15 MP117 6.25 3.15 3.15 3.15 RL 241  1.5 (or 3.34 uM) 1.5 1.5 1.5 WT = wildtype of M.tb EMB as 2HCl salt RL241 as 2HCl salt

TABLE 13 Screening of Substituted Ethylene Diamines Against DrugResistant Patient Isolates - (MIC values in ug/ml) WT 576 587 3806 EMB1.6-1.8 50 50 50 Cmpd#59 0.05 (or 0.13 uM) 0.1 0.05 0.05 Cmpd#109 0.10(or 0.18 uM) 0.2 0.2 0.1 Cmpd#59 as a 2HCl salt Cmpd#109 as a 2CF₃COOHsalt

TABLE 14 Drug Resistance of Each MDR Strain Strain STP STP2 INH1 INH2Rif Emb Eth Kan Cip Cap Cyc 576 W1 R R R R R R R R S R S 587 W R R R R RR S R S S S 3806 W1 R R R R S R R = resistant S = susceptible STP =Streptomycin INH = Isoniazid Rif = Rifampicin Emb = Ethambutol Eth =Ethionamide Kan = Kanamycin Cip = Ciprofloxacin Cap = Capreomycin Cyc =Cycloserine

Example X In Vivo Animal Studies

Animal models were used in the final stages of the drug discovery cycleto assess the anti-microbial efficacy of some substitutedethylanediamine compounds in a representative system of human diseasestate. The in vivo testing approach involves the inoculation of four-sixweek old C57BL/6 mice via aerosol, containing approximately 200 colonyforming units of M. tuberculosis H37Rv.

A. CFU Lung Study

Mice aerosolized with M. tuberculosis H37Rv were examined for 10 to 12weeks following inoculation. Drugs (substituted ethylene diamines) wereadministered via the esophageal cannula (gavage) 7 days/week, startingat either 14 or 21 days post infection. Bacterial load in the lungs offive mice per group were determined at approximately one-week intervalsby viable colony counts. The drugs tested were directly compared to thefront line anti-tuberculosis drug isoniazid, and to the second linedrug, ethambutol. Isoniazid and ethambutol were tested at 25 mg/kg and100 mg/kg, respectively. The substituted ethylene diamines, compound 37,compound 59 and compound 109, were each tested at 1 mg/kg and 10 mg/kg.FIGS. 17 to 19 represent data from three, independent CFU Lung studies.In each study, the number of colony forming units (CFU) that wererecoverable and cultivatable, were determined during various timeintervals (days).

B. Lesion Study

The ability of compound 59 and compound 109 to prevent the developmentof gross pathology due to bacterial burden was determined in conjunctionwith the CFU/Lung Study. The gross pathology was determined by visiblequantitation of lesions on the surface of the lungs. Quantitation byinspection is a good surrogate for CFU determination, and directlycorrelates to the bacterial burden, as determined by the actual colonyforming units. The lesions are first visibly examined, and then thelungs are processed and plated for CFU quantification. The lesion studydemonstrates the ability of the drug to prevent the development of thedisease pathology. FIG. 20 represents data from a lesion study. Thecorresponding CFU results are shown in FIG. 19.

C. Toxicity Study

Toxicity was assessed using a dose escalation study. This study wasperformed with ten C57BL/6 mice per candidate. Every two days, the micewere administered an increased concentration of the drug, and monitoredfor detrimental effects. The administration scheme was 50, 100, 200,400, 600, 800 and 1000 mg/kg. The maximum limit of 1000 kg/mg was basedon the goal of dose escalation, and the solubility of the drugs in thedelivery vehicle. Compound 37 was toxic in mice at 100 kg/mg. Compound59 and compound 109 were tolerated in mice at 1000 mg/kg and 800 mg/kg,respectively.

It should be understood that the foregoing relates only to preferredembodiments of the present invention, and that numerous modifications,or alterations, may be made therein without departing from the spiritand scope of the invention. The entire text of each reference mentionedherein is hereby incorporated, in its entirety, by reference.

Example XI In Vitro Toxicity and Selectivity Indexes for Hit Compounds

Twenty six compounds (including 37, 59 and 109) were tested in an invitro model of toxicity using monkey kidney cells (Vero) and humancervical cancer cells (HeLa) using methods well known to those skilledin the art. The data from this toxicity testing and the MIC data wereused to calculate a selectivity index (SI), the ratio of IC50:MIC (Table15). Selectivity Indexes were ranging from 1.76 to 16.67. Compound 109has the best selectivity index.

TABLE 15 In vitro data for representative compounds Compound MIC (μM)Vero IC50 (μM) SI (IC50:MIC) 66 15.6 28 1.76 40 15.6 25 1.88 41 3.13 192.05 59 15.6 36 2.30 55 15.6 34 2.32 57 11.7 22 2.40 37 7.8 32 4.10 386.25 33 5.28 111 7.81 45 5.76 73 12.5 81 6.48 58 12.5 82 6.56 78 15.6130 8.33 109 1.56 26 16.67

Example XII In Vivo Efficacy of Ethambutol Analogues

Compounds 58, 59, 73, 109, and 111 were selected for in vivo efficacystudies in a mouse model of TB. Compounds 58 and 59 share the samecyclooctyl fragment in their molecules; compounds 58, 73, and 109 shareadamantly moiety, and 109 and 111—the geranyl fragment (FIG. 22).

In these studies, 8-week old inbred female mice C57BL/6 wereintravenously infected with M. tuberculosis. 3 weeks following infectiondrug treatment was initiated (detailed protocol is provided). The drugswere administered orally by gavage. Mice were sacrificed at threetimepoints (15, 30, and 45 days post infection), and CFUs in spleen andlungs were determined (FIGS. 23 and 24). These studies demonstrated thatcompound 109 had activity at doses 1 and 10 mg/kg equal to that ofethambutol at 100 mg/kg

Materials and Methods

Mice. Female C57BL/6 mice of 8 weeks old were purchased from CharlesRiver (Raleigh, N.C.), housed in BSL-2 facility of BIOCAL, Inc.(Rockville, Md.), and were allowed to acclimate at least 4 days priorinfection.

Mycobacteria. An example of frozen and thawed of M. tuberculosis H37RvPasteur was added to 5 ml 7H10 broth medium, with 0.5% BSA and 0.05%Tween 80, incubated 1 week at 37° C., and then 1 ml was added into 25 mlmedium (2-d passage during 2 weeks). Culture was washed twice andresuspended in PBS with 0.5% BSA and 0.05% Tween 80, aliquoted andfrozen at −80° C. To determined CFU of the culture aliquot was thawed,and 10-fold dilutions will be plated on agar 7H9 and CFU count will becalculated 20 days later.

Infection: Frozen sample of culture was thawed, and diluted forconcentration about 10⁶ CFU/ml. Mice were infected with M. tuberculosisH37Rv intravenously through lateral tail vein in corresponded dose in0.2 ml of PBS.

Antimicrobial agents. INH, EMB, Ethambutol analogues.

Protocol of drug treatment: Treatment of mice with compounds wasinitiated 20 days following infection. Compounds were dissolved in 10%ethanol in water and administered by gavage (0.2 ml per mouse). Therapywas given 5 days per week and continued for four or six weeks. Two, fourand six weeks following chemotherapy start mice (6 mice per group) weresacrificed, lungs and spleens were removed and homogenized in sterile in2 ml PBS with 0.05% Tween-80. Homogenates were plated in serialdilutions on 7H10 agar dishes, and incubated at 37° C. CFU counts werecalculated three weeks later.

Statistic analysis. To analyze results of CFUs in organs ANOVA test wasperformed; the significance of the differences was estimated byStudent's test, p<0.05 was considered statistically significant.

Results

In vivo activities of new compounds. The activities of these compoundsare presented in FIGS. 21-24. In the experiment presented in FIGS. 21(spleen) and 22 (lung) mice were infected with 5×10⁵ CFU M. tuberculosisH37Rv and chemotherapy was started 20 days following infection. Micewere treated with INH (25 mg/kg), EMB (100 mg/kg), compounds 73 and 109(both 1 mg/kg and 10 mg/kg). The results indicate that in the spleen,compounds 73 and 109 have activities equal to that of EMB at 100 mg/kg(FIG. 21). In spleen there are no statistical differences betweenactivities of these compounds at 1 mg/kg or 10 mg/kg. In the lung,compound 109 at concentration 10 mg/kg after 4 and 6 weeks was moreeffective than EMB at 100 mg/kg. In the lung, statistically sufficientdifference was shown for compound 109 at concentrations 1 mg/kg and 10mg/kg (FIG. 22). INH was the most active drug in both spleen and lung.

Compounds 73 and 109 were also tested in shorter model with using higherdose of infection (FIGS. 23 and 24). Mice were infected with 5×10⁶ CFUM. tuberculosis H37Rv and chemotherapy was started 15 days followinginfection. Mice were treated with INH (25 mg/kg), EMB (100 mg/kg),compounds 109 (0.1 mg/kg, 10 mg/kg, and 25 mg/kg), 58, 73 and 111 (all25 mg/kg). Mice were treated for 4 weeks. In both the spleen and lung,compound 109 at concentrations 10 mg/kg and 25 mg/kg had activity equalto that of EMB at 100 mg/kg, and at concentration 0.1 mg/kg minimal butsufficient difference with untreated control appeared after 4 weeks oftherapy (FIGS. 23 and 24). Statistically sufficient difference betweencompounds 73 (25 mg/kg) and 109 (25 mg/kg) was detected. In the lungsignificant difference between activities of these compounds was notdetected. Compounds 58 and 111 are active in vivo in both spleen andlung; however, compounds 73 and 109 are preferable. The results of theseexperiments indicate that compounds 73 and 109 in low concentration showactivity equal that of EMB at 100 mg/kg, and in some cases compound 109shows higher activity.

Testing of compounds 111 and 59 was performed in B6 mice infected with5×10⁵ CFU M. tuberculosis H37Rv and beginning chemotherapy 20 daysfollowing infection (FIGS. 25 and 26). Both compounds showedanti-tuberculosis activity at concentration 10 mg/kg comparable to thatof EMB at 100 mg/kg.

In all experiments, INH showed higher activity than EMB and othercompounds decreasing load of bacteria in organs on 2-3 logs during 4-6weeks of chemotherapy; new compounds similar to EMB (100 mg/kg)decreased load of bacteria on 1.0-2.0 logs. Among studied compounds 73and 109 are the most preferable, because the highest capacity todecrease mycobacteria in organs and its parameters of toxicity andpharmacology kinetics.

Example XIII In Vivo Toxicity

Preliminary dose acceleration studies in mice have indicated thatcompound 109 can be well tolerated at doses up to 800 mg/kg and compound59 up to 1000 mg/kg. Compound 37 was fatal at doses 100 mg/kg (ClifBarry, NIAID, unpublished results).

Compound 109 was mostly used in the form of dihydrochloride at fivedifferent doses, and 37—solely as hydrochloride salt at two doses.

Mice were given a one-time dose of the compounds at concentrations 100,300 or 1000 mg/kg using the gavage method. Each dose of each compoundconsisted of one group of 3 mice. Monitoring of the mice was done twicea day for the duration of the experiment. Mice surviving one weekpost-drug administration were sacrificed; critical organs wereaseptically removed and observed for abnormalities and evidence of drugtoxicity. The MTD (mg/kg) is the highest dose that results in nolethality/tissue abnormality.

Methods:

1. Treatment of mice: C57BL/6 female mice (6-8 weeks in age) are given aone-time dose of the compound at concentrations 100, 300 or 1000 mg/kgusing the gavage method. The compounds are dissolved in the appropriateconcentration of ethanol in distilled water and administered in a volumeof 0.2 ml per mouse.2. Observation of mice: Mice will be observed 4 and 6 hours postadministration, then twice daily for one week. Survival and body weightof mice will be closely monitored throughout the study.3. Assessment of drug toxicity: Mice exhibiting signs of any abnormalappearance or behavior or those remaining in a group in which other micedid not survive to day 7 will be sacrificed for assessment of drugtoxicity. Critical organs will be aseptically removed and observed;tissues from the liver, heart, and kidneys are extracted and placed into10% formalin solution. These fixed tissues are sectioned and examinedfor abnormalities resulting from drug toxicity.

These studies indicate that the maximum tolerated dose for the compound109 is 600 mg/kg (Table 16). No visible changes in organs were observed.Dose 800 mg/kg was fatal: out of a group of 3 mice, two animals diedwithin 3 days (Table 17). Compound 37 was well tolerated at doses 100and 300 mg/kg. No visible changes in organs were observed. Additionalexperiments to evaluate maximum tolerated dose and in vivo efficacy forthe compound 37 are being conducted.

TABLE 16 Determination of a maximum tolerated dose for the compounds 109and 37 in mice. 109 at 109 at 109 at 109 at 37 at 100 mg/kg 300 mg/kg600 mg/kg 1000 mg/kg 100 mg/kg Day of Day of Day of Day of Day of DayMice death Mice death Mice death Mice death Mice death Apr. 08, 2003 1 33 3 3 2-4 h 1 Apr. 09, 2003 2 3 3 3 2 2 2 Apr. 10, 2003 3 3 3 3 2 2 Apr.11, 2003 4 3 3 3 1 4 2 Apr. 13, 2003 6 3 3 3 0 6 2 Apr. 14, 2003 7 3 3 3— 2

TABLE 17 Determination of a maximum tolerated dose for the compounds 109And 37 in mice 109 as HCl 109 as TFA 37 at 37 at salt at salt at 100mg/kg 300 mg/kg 800 mg/kg 800 mg/kg Day of Day of Day of Day of Date DayMice death Mice death Mice death Mice death Apr. 29, 2003 1 3 3 3 1 Apr.30, 2003 2 3 3 2/1 2 1 May 01, 2003 3 3 3 1/1 3 1 May 02, 2003 4 3 3 1 105.03. 5 3 3 1 1 05.04 6 3 3 1 1 May 05, 2003 7 3 3 1 1

Example XIV Pharmacokinetic Studies of the Compounds 37, 59, and 109

Initially, analytical methods for determination of the compounds hadbeen developed that allowed to carry out all the PK experiments, secFIG. 29. Here is a brief description of the experiment: (1) plasmaspiked with tested compounds and 10 uL of Terfenadine or plasma samples(200 uL) added; (2) ACN (2 mL) added to precipitate protein and spin at2,500 rpm; (3) evaporate supernatant to dryness; (4) add 200 uL of thediluting solvent: methanol (with 0.1% of trifluoroacetic acid): ammoniumacetate (80/20); (5) vortex, spin, and use supernatant; (6) run LC/MS/MSon Sciex API 3000.

Biostability studies of the compounds in plasma were carried out usingconcentrations 1 and 15 mg/ml. The compounds were incubated for 1, 2, 3& 6 hr at 37° C. (Table 18). In addition, it was found that all testedcompounds were stable in plasma at 24° C., pH 2 and 7.4 up to 24 hr.

TABLE 18 Biostability of tested compounds in plasma. Comp. Human Dog RatMouse 37 20% ↓ stable 35% ↓ stable 59 stable stable stable stable 10930% ↓ 40% ↓ stable stablePilot PK study of the compounds 37, 59, and 109 in mice was conductedusing a cassette dosing: all the three analogs were formulated togetherin saline at 1.5 mg/mL, and administered to mice simultaneously orallyat 25 mg/kg, peritoneally at 6 mg/kg, and intravenously. It was foundthat doses 15 and 7.5 mg/kg caused death of mice, 3.75 mg/kg appearedlethargic immediately after dosing but then appeared normal appearance afew minutes later; 3 mg/kg displayed no adverse reactions and hence wasused as intravenous dose. Obtained data are presented on FIGS. 30, 31,and 32 (tested compounds were studied under the NCI′ indexation NSC) andsummarized in Table 19.

TABLE 19 PK Parameters of tested compounds 37, 59, and 109 after acassette dosing to mice. Route i.v. i.v. i.v. i.p. i.p. i.p. p.o. p.o.p.o. Compounds 37 59 109 37 59 109 37 59 109 Dose (mg/kg) 3 3 3 6 6 6 2525 25 AUC(ng · h/mL) 954 384 1006 1372 272 1099 1602 169 655 Cmax(ng/mL) 970 296 1192 630 217 935 263 28.7 227 T½ (h) 4.8 6.4 5.5 4.9 9.74.4 N/A N/A N/A CL (mL/kg/h) 3530 8043 3240 Bioavailability (%) 72 35 553.3 0.9 2.7 Urine excretion (%) .71 1.9 .92 <0.01 <0.01 <0.01 N/A N/AN/A N/A—not detectable.

Conducted pharmacokinetic studies indicated that compound 59 (NSC 722040by the NCI index) has relatively poor PK profiling (AUC, Cmax) andfurther testing of this compound was abandoned. Based on preliminarytoxicity data compound 37 was also ruled out as possible candidate.Therefore, compound 109 (NSC 722041 by the NCI) was selected for furtherPK analyses.

It has been shown that compound SQ 109 reaches and exceeds its MinimumBactericidal Concentration MBC (313 ng/ml) in plasma when administeredeither iv or intravenously orally (p.o.), has a half-life of 5.2 h, andhas total clearance less than hepatic blood flow (FIG. 33, Table 20).

TABLE 20 Pharmacokinetic parameters of the compound 109 Parameters i.v.p.o. Dose (mg/kg) 3 25 AUC (ng · h/mL) 792 254 T_(1/2 el) (h) 3.5 5.2C_(max)(ng/mL) 1038 135 T_(max)(h) 0 0.31 CL (mL/kg/h) 3788 Vd_(ss)(mL/kg) 11826 Bioavailability 3.8

Its oral bioavailability is only 3.8% when administered p.o but this isexplained by its unique tissue distribution pattern. Tissue distributionstudies have demonstrated that SQ109 primarily distributes into thelungs and spleen (FIGS. 34 and 35), which is highly advantageous for ainfection that characteristically manifests as a lung disease.

By using an ultracentrifugation method, it was found that plasma proteinbinding of the compound 109 is concentration dependent and varies from15% (20 ng/ml) to 74% (200 ng/ml) to 48% (2000 ng/ml). After i.v. dosing(3 mg/kg) the compound distributes between plasma and red blood cells ina ratio 70.6:29.4.

Little is known of the fate of the compound in the body, since the totalamount of the compound after excretion (urine and feces) does not exceed3% of the delivered dose (Table 2).

TABLE 21 Amounts of the compound 109 cumulatively excreted in mouseurine and feces following single administration Period after dosing (h)Dose/ Total Route Samples 0-4 4-8 8-24 24-32 0-32 3 mg/kg Urine <0.01<0.01 0.03 0.01 0.04 i.v. Feces <0.01 0.01 0.04 <0.01 0.06 25 mg/kgUrine — — — — p.o. Feces 0.48 0.31 1.12 0.08 2.0

Initial attempts to identify metabolites of the compound 109 in urine,did not provide evidence of breakdown products, FIG. 36. For example,there was no evidence for the formation of conjugated metabolites (M⁺521) in the mouse urine during first 24 hr following compound'sadministration, FIG. 37. Conjugated metabolites are products of thetypical metabolic pathway N-glucoronidation formed by reaction withglucuronic acid (D. A. Williams and T. L. Lemke in Foye's Principals ofMedicinal Chemistry, 5^(th) Ed., p.202).

Example XV In vitro Pharmacokinctic Studies of Compound 109

In vitro Pharmacology and early ADMET (Absorption, Distribution,Metabolism, Excretion, Toxicity) studies of the compound 109 werecontracted out to CEREP (15318 NE 95^(th) Street, Redmond, Wash. 98052,USA, www.cerep.com, tel 425 895 8666) under a Service Agreement andincluded testing against 30 standard receptors (see CEREP Tables 22 and23, provided in FIGS. 38 and 39, five CYP450 enzymes, hERG (K+ channel),aqueous solubility, predicted intestinal permeability, and metabolicstability (data presented in FIG. 40 Tables 24(a-m)).

Example XVI Bis(2-Adamantyl)ethylenediamine, SQBisAd

Compounds with the best Selectivity Indexes, such as 109, 58, 73, 78,(Table 15) and good in vivo data share the same adamantane fragment(FIG. 20). A compound that would have solely this fragment (on bothsides of the ethylene linker) was contemplated. During preparation oftargeted 100,000 compound library of ethambutol analogues, 70,000compounds were proven to be formed, but 30,000 were failures. Thisparticular compound was not initially detected perhaps because it wassynthesized in very low yield or because it was never made due to stericfactors.

In the synthetic scheme used for preparation of the library Scheme 1(FIG. 41), sterically hindered amines on the second step rarely gaveproducts. Analyzing MS data for a number of original plates it can bestated that 2-adamantanamine when used as R₁NH₂ seldom yield desirableproducts and this can be explained because of existence of stericallyhindered reaction site on the step 2 or step 3 of the synthesis Scheme 2(FIG. 41).

Compound SQBisAd can be prepared by “wet chemistry” using the sameroute, Scheme 3 (FIG. 41), it is documented that 2-adamantamine (used ascommercially available hydrochloride) does provide products when used onthe 1 and 2 steps. Due to the symmetrical nature, this compound can besynthesized by alternative routes. We have prepared SQBisAd by reductivealkylation of ethylnediamine by 2-adamantanone using sodiumcyanoborohydride. Final product (without additional purification)demonstrated MIC (Minimal Inhibitory Concentration) equal or better thancompound 109.

Example VIII Generating the Diamine Library with a Modified Linker

General Methods All reagents were purchased from Sigma-Aldrich. Rinkacid resin was purchased from NovaBiochem, Inc. Solvents acetonitrile,dichloromethane, dimethylformamide, ethylene dichloride, methanol, andtetrahydrofuran were purchased from Aldrich and used as received. Solidphase syntheses were performed on Quest 210 Synthesizer (ArgonautTechnologies) and combinatorial chemistry equipment (WhatmanPolyfiltronics and Robbins Scientific). Evaporation of the solvents wasdone using SpeedVac AES (Savant). Mass spectra data were obtained byElectrospray Ionization technique on Perkin Elmer/Sciex, API-300, TQMSwith an autosampler.

The activation of the Rink-resin, the addition of the amine, and theacylation step were carried out in 10 ml tubes using the Quest 210Synthesizer. Removal of the FMOC group, reductive alkylation reactionwith carbonyl compounds, the reduction with Red-Al, and the cleavagefrom the solid support were carried out in 96-deep (2 ml) well,chemically resistant plates.

Step 1. Activation of the Rink-Acid Resin.

A suspension of the Rink-acid resin (coverage of 0.43-0.63 mmol/g), 6 g(up to 3.78 mmol), in 80 ml of 2:1 mixture of dichloromethane and THFwas distributed into 20 tubes, 4 ml per tube, filtered and washed twicewith THF. A solution of triphenylphosphine (5.7 g, 21.75 mmol) in 40 mlof THF was added, 2 ml/tube, followed by the addition of a solution ofhexachloroethane (5.09 g, 21.45 mmol) in 20 ml of THF, 1 ml/tube. After6 h the resins were washed with THF (2×4 ml) and dichloromethane (2×4ml).

Step 2. Addition of the First Amine.

Each tube was charged with 3 ml of dichloroethane, EtNiPr₂, (0.2 ml,1.15 mmol), and the corresponding amine (1 mmol). (When a selected aminewas a solid, it was added as a solution or a suspension in DMF).Dichloroethane was added to each tube to fill up the volume 4 ml. Thereaction was carried for 8 h at 45° C. and 6-8 h at room temperature.The resins were filtered, washed with a 2:1 mixture of dichloromethaneand methanol (1×4 ml), then with methanol (2×4 ml), and suck dry.

Step 3. Acylation with Fmoc Protected Amino Acid.

The resins were pre-washed with dichloromethane (2×4 ml). Each tube wascharged with 2 ml of dichloromethane, HATU (2 mol excess to loadedresin, 0.14 g, 0.39 mmol, dissolved in 1 ml of DMF), and 0.47 mmol (2.5mol excess to loaded resin) of amino acid dissolved in 1 ml of DMF, andallowed to stir for 8 h at 45° C. and 6-8 h at room temperature. After16 h the resins were filtered, washed with 1:1 mixture of DMF anddichloromethane (1×3 ml), dichloromethane (1×3 ml) and acylation wasrepeated with the same amount of reagents. At the end, the resins werefiltered, washed with 1:1 mixture of DMF and dichloromethane (1×3 ml),and methanol (3×3 ml), sucked dry (on Quest) for 30 min and transferredinto vials (one resin per vial), and dried in a desiccator under vacuumfor 1 h. After this step all resins were subjected for quality controlusing MS spectra.

Step 4. Alkylation of the Amino Group.

Deprotection. Ten prepared resins from the first three steps were pooledtogether, leaving approximately 0.05 g of each in the individual vialsfor all necessary deconvolutions. A suspension of the resin mixture(2.0-2.5 g) in 100 ml of a 2:1 mixture of dichloromethane and THF wasdistributed into two 96-well filterplates and filtered using afiltration manifold. The reaction plates were transferred intocombiclamps, and 0.2 ml of 20% solution of piperidine in DMF was addedto remove Fmoc protecting group and allowed to stay for 10 min. After 10min plate was filtered, washed with 0.2 ml of DMF, and deprotection wasrepeated with 0.2 ml of 20% solution of piperidine in DMF and allowed tostay for 20 min. After that plate was filtered, washed with DMF (0.2 mlper well) and dichloromethane (2×0.5 ml per well).

Reaction with the carbonyl compounds. Each well in row A on the reactionplate was charged with 0.1 ml of dichloromethane, 0.08 ml of ˜1.0Msolution of appropriate acid in DMF from master plate, 0.05 ml DMFsolution of PyBrop, (0.015 g, 0.03 mmol, 2.5 mol excess to loaded resin)and 0.05 ml of EtNiPr₂ in dichloromethane (0.022 ml, 0.13 mmol, 10 molexcess to loaded resin). Each well in rows B through H was charged with0.1 ml of THF, 0.160 ml of 1.0 M solution of appropriate aldehyde orketone in DMF from master plate and allowed to react for 30 min. After30 min 0-075 ml (0.075 mmol) of 1.0 M solution of NaBCNH₃ were added.The reaction plates were sealed and kept at RT for 72 h. At the end, theresins were filtered, washed with THF, DCM (1×1 ml), methanol (2×1 ml)and dried in desiccator under vacuum for 2 h.

Step 5. Reduction with Red-Al.

The reaction plates were placed into combiclamps. A 1:6 mixture ofRed-Al (65+w % in toluene) and THF was added, 0.6 ml per well (0.28 mmolof Red-Al per well), and allowed to react for 4 h. After the reactioncompletion the resins were filtered, washed with THF (2×1 ml), methanol(3×1 ml) and dried in the filtration manifold.

Step 6. Cleavage.

This step was carried out using a cleavage manifold. The reaction plates(placed on the top of the collection plates in this manifold) werecharged with a 10:85:5 mixture of TFA, dichloromethane, and methanol,0.5 ml per well. After 15 min, the solutions were filtered and collectedinto proper wells of the collection plates. The procedure was repeated.Solvents were evaporated on a speedvac, and the residual samples wereready for testing.

Deconvolution Example.

Deconvolution of the active wells was performed by re-synthesis ofdiscrete compounds, from the archived FMOC-protected a-aminoacetamideresins (10 resins, 0.05-0.10 g each), which were set aside at the end ofthe acylation step before the pooling. Each resin was assigned adiscrete column (1, or 2, or 3, etc.) in a 96-well filterplate, and wasdivided between X rows (A, B, C, etc), where X is the number of hitsdiscovered in the original screening plate. To each well, in a row, aselected carbonyl compound (present in the hit) was added along withother required reagents: the first selected carbonyl compound was addedto the resins in the row “A”, the second carbonyl compound—to the resinsin the row “B”, the third carbonyl compound—to the resins in the row“C”, etc. A lay-out of a representative 96-well deconvolution plate isshown in Table 28, FIG. 52.

The reaction plates were sealed and kept at RT for 72 h. At the end, theresins were filtered, washed with THF, DCM (1×1 ml), methanol (2×1 ml)and dried in desiccator under vacuum for 2 h. Reduction and cleavagewere performed according to steps 5 and 6 of the synthetic protocol. Theproduct wells from the cleavage were analyzed by ESI-MS (ElectrosprayIonization Mass Spectroscopy) to ensure the identity of the actives, andwere tested in the MIC assay. A summary of the ESI-MS data is providedbelow. A list of compound hits and structures is provided in Table 30,FIG. 53.

Compound 673

N²-[(2-methoxy-1-naphthyl)methyl]-3-phenyl-N′-(3-phenylpropyl)propane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺439.2

Compound 674

N²-[2-(benzyloxy)ethyl]-N′-(3,3-diphenylpropyl)-4-(methylthio)butane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺463.4.

Compound 675

N¹-(3,3-diphenylpropyl)-4-(methylthio)-N²-(3-phenylpropyl)butane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺447.2

Compound 676

N²-(cyclohexylmethyl)-N¹-(3,3-diphenylpropyl)-4-(methylthio)butane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺425.1

Compound 677

N¹-(3,3-diphenylpropyl)-N²-(2-ethoxybenzyl)-4-(methylthio)butane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺463.1

Compound 678

N²-[2-(benzyloxy)ethyl]-N¹-[(6,6-dimethylbicyclo[3.1.1]hept-2-yl)methyl]-4-(methylthio)butane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺405.3

Compound 679

N′-[(6,6-dimethylbicyclo[3.1.1]hept-2-yl)methyl]-4-(methylthio)-N²-(3-phenylpropyl)butane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺389.5

Compound 680

N²-(2-chloro-4-fluorobenzyl)-4-methyl-N′-(4-methylbenzyl)pentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺363.3, 365.5; (MCH₃CN) 403.3, 405.3.

Compound 681.

N²-[2-(benzyloxy)ethyl]-N¹-[2-(4-methoxyphenyl)ethyl]-4-methylpentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺385.1.

Compound 682.

N²-[3-(4-chlorophenoxy)benzyl]-N¹-[2-(4-methoxyphenyl)ethyl]-4-methylpentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺467.1, 469.2.

Compound 683.

N²-(4-isopropylbenzyl)-N¹-[2-(4-methoxyphenyl)ethyl]-4-methylpentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺383.3

Compound 684.

N¹-[2-(4-methoxyphenyl)ethyl]-4-methyl-N²-[(2E)-3-phenylprop-2-enyl]pentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺367.3; [M-(CH₂CH═CHPh)₂H]+ 251.

Compound 685

N²-[2-(benzyloxy)ethyl]-4-methyl-N¹-(3-phenylpropyl)pentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺369.1.

Compound 686.

N²-(2-chloro-4-fluorobenzyl)-4-methyl-N′-(3-phenylpropyl)pentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺377.2, 378.9.

Compound 687.

N²-[3-(4-chlorophenoxy)benzyl]-4-methyl-N′-(3-phenylpropyl)pentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺451.1, 453.3.

Compound 688.

N²-(4-isopropylbenzyl)-4-methyl-N′-(3-phenylpropyl)pentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺367.3.

Compound 689

4-methyl-N²-[(2E)-3-phenylprop-2-enyl]-N′-(3-phenylpropyl)pentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺351.2.

Compound 690

N²-(2-ethoxybenzyl)-4-methyl-N′-(3-phenylpropyl)pentane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺369.1.

Compound 691.

N²-decahydronaphthalen-2-yl-N′-[2-(4-fluorophenyl)ethyl]-3-thien-3-ylpropane-1,2-diamine.Mass spectrum (ESI) m/z (MH)⁺415.3.

Example XIX In Vitro Activity of Rifampicin with Compound 109 orIsoniazid

Compound 109 demonstrated potent in vitro and in vivo killing of M.tuberculosis as an individual compound. Herein, are examples providing amulti-drug regime to evaluate the effects of the compounds of table 3 oninhibition of bacterial growth in vitro and in mouse models oftuberculosis when used in combination with standard tuberculosis drugs.Rifampicin, compound 109 and isoniazid were selected for in vitroactivity (FIG. 54). The study was carried out using BACTEC growthkinetics. MIC for RIF was 0.2 ug/ml, SQ109 (compound 109) 0.32 ug/ml,and INH 0.025 ug/ml. These studies demonstrated that compound 109 incombination with Rifampicin suppresses the growth index over the lengthof study. The growth suppression of Rif and compound 109 is achievedeven when the MIC concentration of compound 109 is 1/10^(th) and1/20^(th) MIC. Clearly, the combination of Rif and compound 109 issuperior to Rif alone, or Rif and INH. Compound 109 at 0.5 MIC inhibitedgreater than 99% growth of M. tuberculosis (H37Rv) inoculum when used incombination with as low as 0.1 MIC RIF. The x/y quotient value was 0.29,indicating synergistic drug action. This synergy was also seen when 0.5MIC RIF was used in combination with 0.05, 0.1, and 0.2 MIC SQ109, withcorresponding x/y quotient values of 0.32, 0.16, 0.4, respectively. Theresults indicate synergistic activity for growth inhibition of M.tuberculosis by the combination of RIF and SQ 109.

Example XX In Vivo Activity of Compound 109 with Standard TuberculosisDrugs

A rapid in vivo model of TB where infected animal weight loss is theindicator for tuberculosis disease progression was used to elevatednovel compounds and combinational therapies. Rifampicin, INH, EMB, PZA,Moxi (standard tuberculosis drugs) and compound 109 were selected for invivo studies in mice (FIG. 55). These studies demonstrated that compound109 in combination with one or more standard tuberculosis drugsmodulates mice body weight and is an indicator of drug efficacy. In thisstudy, Rifampicin and compound 109, or INH and compound 109, achievedbody weights that were comparable to non-infected controls. Conversely,Moxi and compound 109, or EMB and compound 109, resulted in body weightsthat were closer to placebo treated controls. Clearly, the combinationof Rifampicin and compound 109 is superior to Rifampicin or INH alone.

Example XXI Rapid Model, In Vivo Activity of Compound 109 AgainstStandard Tuberculosis Drugs

In this example Rifampicin and compound 109 were selected for in vivostudies in mice (FIG. 56A and FIG. 56B). In this study, Rifampicin andcompound 109 achieved body weights that were comparable to non-infectedcontrols. Conversely, compound 109, or rifampicin alone resulted in bodyweights that were closer to placebo treated controls over the course ofchemotherapy. Clearly, the combination of Rifampicin and compound 109 issuperior to Rifampicin or compound 109 alone.

Example XXII In Vivo Activity of Combination Therapy

A combination therapy of SQ109 (compound 109) at 10 mg/kg and RIF at 2mg/kg given orally was more efficacious in preventing body weight lossin infected mice than either single drug therapy given at the same dose(FIG. 57). By the 3 wk of chemotherapy, average body weight of 6 micethat received combination RIF+SQ109 therapy was 24.3 grams andindistinguishable from the 24.5 g of the uninfected control group. Incomparison, mice receiving SQ109 or RIF alone at the corresponding doseshad average weights of 21.3 g and 19.7 g, respectively: the averageweight for infected mice not treated with drugs was 17 g.

Example XXIII In Vivo Activity of Combination Therapy (Standard ChronicModel)

The in vivo enhanced activity of combination treatment was confirmedthrough the use of a standard chronic mouse model of tuberculosis (FIG.58). During 4 week therapy in the chronic TB model, SQ109 by itselfreduced CFU in lung from 8.1 log₁₀CFU (control untreated animals) to 6.6log₁₀CFU; RIF (20 mg/kg) by itself reduced CFU to 5.8 log₁₀; but thecombination of SQ109+RIF (given at their most efficacious doses 10 and20 mg/kg respectively) reduced CFU to 4.8 log₁₀, an additional log₁₀CFUlower than either drug alone. Combination of INH+RIF+SQ109 (at 25, 20and 10 mg/kg) achieved the same CFU at wk 3 as INH+RIF+EMB (at 25, 20and 100 mg/kg) at wk 4, 1 wk earlier than standard drug combination(FIG. 59). By wk 4 the SQ109 combination was more effective by a halflog₁₀ (3.2 log₁₀CFU) than EMB combination therapy (3.7 log₁₀CFU). Thedifference in CFU reduction was statistically significant. Compound 109also enhanced in vivo killing of M. tuberculosis in TB mouse models whenused in combination with RIF alone or RIF+INH. On the basis of theseresults, it is proposed that the synergistic activity of compound 109with RIF suggests that it could replace ethambutol in the intensivephase of TB therapy with the 3- or 4-drug combination. Further, compoundSQ 109 may provide additional benefit when combined with RIF in thecontinuation phase, because it has potent activity by itself. Moreover,given that the mode of action is distinct from RIF, this aspect willassist RIF from the emergence of resistant organisms, whilesimultaneously providing a synergistically enhanced activity.

Example XXIV In Vitro Pharmacokinetic Studies of Compound 109

In vitro testing for safety pharmacology and early ADMET (Absorption,Distribution, Metabolism, Excretion, Toxicity) studies of compound 109were contracted out to CEREP (15318 NE 95^(th) Street, Redmond, Wash.,98052, USA www.cerep.com) under a service agreement and includedevaluation of inhibitory binding to a panel of 27 standard receptors andthree transporters (See FIG. 60 (Table 31) and FIG. 61 (Table 32)) whichincluded: adenosine receptors (A1 and A2A), adrenergic receptors (alpha1, alpha 2, beta 1), angiotensin II receptor (AT1), benzodiazapenereceptor (BZD), bradykinin receptor (B2), cholecystokinin receptor(CCK1), dopamine receptors (D1, D2S), endothelin receptor (ETA), GABAreceptor (GABA), glutamate receptor (NMDA), histamine receptor (H1central), melanocortin receptor (MC4), muscarinic receptor (M,non-selective), neurokinin receptor (NK1), neuropeptide Y receptor (Y),nicotinic receptor (neuronal, alpha-BGTX-insensitive), opiate receptor(non-selective opiate), orphanin receptor (ORL1), phencyclidine receptor(PCP), serotonin receptor (5-HT), sigma receptor (sigma non-selective),steroid receptor (glucocorticoid receptor, GR), and NE, DA, and 5-HTtransporters.

The specific ligand binding to the receptors is defined as thedifference between the total binding and the nonspecific bindingdetermined in the presence of an excess of unlabelled ligand. Compound109 (SQ109) was tested at 10 μM, and the results were expressed as apercent of control specific binding (Table 31) or as a percent ofinhibition of control specific binding (Table 32) obtained in thepresence of SQ109.

In summary it can be seen that greater than 50% inhibition was observedfor D1 and D2S dopamine receptors (51% and 73%). Similarly, verysignificant inhibition was observed for melanocortin MC4 receptor (90%)which is known to exert a large influence on food intake. Furthermore, avery significant inhibition was observed for muscarinic M receptors(96%).

Opiate receptors which control pain, immune responses, and functions andare linked to effects of morphine and heroin were observed to display a58% inhibition of control specific binding. Sigma receptors that havebeen shown to play an important role in antidepressive effects alsodemonstrated a high degree of inhibition (106%). Norepinephrine NEtransporter plays an important role in the pathophysiology of depressionand in the mechanism of action of antidepressant drugs was observed tohave 87% inhibition of control specific binding. Another transporter,Dopamine DA transporter linked to substance abuse and attention deficithyperactivity disorder (ADHD) was observed to display 63% inhibition.Serotonin 5-HT transporter implicated in the etiology of several diseasestates including, but not limited to, mental illnesses, for example,depression, anxiety, schizophrenia, eating disorders, migraines,obsessive compulsive disorder, and panic disorder were also observed todisplay a 95% inhibition of control specific binding. While not wishingto be bound by the following theory, it is believed that compoundscomprising a Ph-ethyleneamine component, including but not limited to,compound 73 also share CNS activity.

Example XXV Spectrum of Activity Testing—Aerobic and Anaerobic Bacteria

Compound 109 was tested against a representative panel of commonlyencountered clinical microorganisms comprising opportunistic pathogens,target pathogens and normal human flora. The spectrum of activity ofcompound 109 was evaluated by performing antimicrobial susceptibilitytesting against a collection of aerobic and anaerobic bacteria, fungi,and mycobacteria. MICs for all organisms were established using theappropriate NCCLS (National Committee for Clinical Laboratory Standards)recommended standard methods and quality control strains.

Compound 109 displayed activity against gram-positive aerobes. Inparticular, compound 109 (SQ-109) demonstrated the best activity againstStreptococcus pneumoniae with MICs ranging from 4-8 ug/ml. MICs for allspecies of enterococci tested ranged from 16-64 ug/ml while MICs for allStaphylococci tested ranged from 16-32 ug/ml (FIG. 62, Table 33).

Compound 109 displayed activity against gram-negative aerobes. SQ-109demonstrated limited activity against all enterobacteriaceae tested withMICs ranging from 32->64 ug/ml with slightly lower MICs seen among thenon-enterobacteriaceae (16->64 ug/ml). MICs for three Haemophilusinfluenzae isolates tested ranged from 1-32 ug/ml. SQ-109 demonstratedthe best activity when tested against Helicobacter pylori with an MIC of4 ug/ml for all three strains tested (FIG. 63A and FIG. 63B (Table 34)).

Compound 109 displayed activity against anaerobes. SQ-109 MICs rangedfrom 16-64 ug/ml for anaerobes Propionibacterium acnes, Bacteroidesfragilis, and Clostridium difficile (see FIG. 64, Table 35).

Example XXVI Spectrum of Activity Testing—Fungi

Compound 109 was tested against a representative panel of commonlyencountered clinical microorganisms comprising opportunistic pathogens,target pathogens and normal human flora. The spectrum of activity ofcompound 109 was evaluated by performing recommended broth microdilutionmethods. NCCLS M27-A2, 2003 for yeast and NCCLS M38-A, 2003 for mold.

SQ-109 demonstrated good activity against Candida albicans with MICsranging from 4-8 ug/ml. Additionally, Mold MICs for three isolates ofAspergillus fumigatus tested were 16 ug/ml (FIG. 65, Table 36).

Example XXVII Spectrum of Activity Testing—Mycobacteria

Compound 109 was tested against a representative panel of commonlyencountered clinical microorganisms comprising opportunistic pathogens,target pathogens and normal human flora. The spectrum of activity ofcompound 109 was performed by using the BACTEC 460 TB system (BectonDickinson, Cockeysville, Md., USA) according to the manufacturersinstructions. MICs for MOTT were determined suing the agar proportionmethod recommended for testing slow-growing mycobacterium species, whileMICs for the rapid-grower mycobacteria were performed suing therecommended broth microdilution method (NCCLS M24-A, 2003).

SQ-109 demonstrated very good activity when tested against M.tuberculosis [MTB] (0.25-0.5 ug/ml), M. bovis (0.25 ug/ml), and M. bovisBCG (0.5 ug/ml). Among resistant MTB strains tested; SQ-109 had an MICof 0.25 ug/ml for an INH-resistant MTB strain, and an MIC of 0.5 ug/mlagainst an EMB-resistant strain (Table 37).

SQ-109 showed less activity against Mycobacteria-other-than-TB (MOTT)with an MIC of 8 ug/ml for three M. marinum strains tested, an MIC of 16ug/ml for three M. kansasii tested, and MICs ranging from 8-32 ug/ml forthree M. avium complex (MAC) isolates examined (Table 38).

SQ-109 showed good activity against rapid-grower M. fortuitum with anMIC of 1 ug/ml for all strains tested and less activity against the moreresistant members of the M. chelonae group (M. chelonae and M.abscessus) with an MIC of 16 ug/ml for three strains tested (FIG. 66,Table 39).

The results of examples XXV-XXVII demonstrate the best inhibitoryactivity against several species of mycobacterium in the MTB complex (M.tuberculosis, M. bovis, and M. bovis BCG), Mycobacterium-forluitum,Mycobacterium marinum, Helicobacter pylori, Streptococcus pneumoniae andCandida albicans. SQ-109 was also found to be equally active againstsusceptible and resistant strains of M. tuberculosis.

Example XXVII Synergistic Interactions of SQ109 with Front-LineAntitubercular Drugs In Vitro Summary

The purpose of this study was to determine interactions of SQ109 withexisting antitubercular drugs in vitro and assess its potential toimprove combination drug activities against Mycobacterium tuberculosis.

Two-drug combinations at various concentrations below their minimalinhibitory concentrations (MIC) were tested for growth inhibition of M.tuberculosis using the BACTEC 460 system in vitro. Drug interactionswere evaluated based on the quotient values that were derivednumerically from the growth indices of cultures treated with singleantibiotics or combination treatment with two antibiotics.

SQ 109 at 0.5 its MIC demonstrated strong synergistic activity with 0.5MIC isoniazid and as low as 0.1 MIC rifampicin in inhibition of M.tuberculosis growth. Additive effects were observed between SQ109 andstreptomycin, but neither synergy nor additive effects were observedwith the combination of SQ109 with ethambutol or pyrazinamide. Thesynergy between SQ109 and rifampicin was also demonstrated usingrifampicin-resistant (RIF^(R)) M. tuberculosis strains, SQ109 loweredthe MIC of rifampicin against these drug-resistant strains.

SQ109 interacts synergistically with isoniazid and rifampicin, two ofthe most important front-line TB drugs.

Materials and Methods

Antimicrobial drugs isoniazid, rifampicin, streptomycin, and ethambutolwere purchased from Sigma-Aldrich. St. Louis, Mo. Stock solutions ofisoniazid, streptomycin, and ethambutol were prepared in distilled anddeionized water at 10 mg/mL, sterilized by filtration, and stored frozenat −80° C. Stock solutions of rifampicin, at 1 or 10 mg/mL, wereprepared in methanol and stored at −80° C. Pyrazinamide was purchased asthe drug reconstituting kit from Becton Dickinson, Cockeysville, Md.,and a stock solution was prepared following instructions by themanufacturer. Stock solutions of SQ109 were prepared in methanol at 1mg/mL and stored at 80° C.

M. tuberculosis Strains:

M. tuberculosis strain H37Rv was the same strain used in previousstudies documenting activity of chemical compounds in our library. Themono RIF^(R) M. tuberculosis clinical isolates used in this study wereobtained from the Department of Health and Mental Hygiene, CentralLaboratory, State of Maryland. The drug susceptibility profiles ofstrains 3185 and 2482 were previously determined at the state laboratoryand later confirmed at Sequella. Both strains were susceptible to 0.1mg/L isoniazid, 2.5 mg/L ethambutol, and 2 mg/L streptomycin. RifampicinMIC was 24 mg/L for strain 3185 and >100 mg/L for strain 2482. Thenature of rpoB mutation associated with rifampicin resistance was notdetermined.

Media Middlebrook 7H11 agar (Difco, Becton Dickinson) supplemented with10% OADC (Difco) was used to grow M. tuberculosis for BACTEC inocula.BACTEC 71112B medium (Becton Dickinson) was used for all drugcombination experiments except for pyrazinamide+SQ109: this combinationwas evaluated in PZA Test Medium (Becton Dickinson).

Assessment of drug effects on M. tuberculosis growth BACTEC 460 system(Becton Dickinson) was used to determine MIC for each individual drugand to study the combined effects of drugs on M. tuberculosis growth invitro. M. tuberculosis H37Rv or RIF R M. tuberculosis isolates weregrown on 7H11 agar plates. Bacterial inocula were prepared from 4 to 6week old plates by transferring loopfuls of bacteria into capped glasstubes containing dilution fluid and glass beads and vortexing to breakthe clumps. Suspensions of M. tuberculosis at 1 McFarland Standard, freeof clumps, were inoculated into Middlebrook 7H12 medium vials containingvarious combinations of test drugs (SQ109, isoniazid, rifampicin,ethambutol, streptomycin). The growth of the bacilli, expressed as thegrowth index or GI, was monitored daily by measuring the release of¹⁴CO₂ after the bacteria consumed ¹⁴C labeled palmitic acid in themedia. To determine the combined effects of pyrazinamide and SQ109, weused the special pyrazinamide test medium from Becton Dickinson. No-drugcontrols, including the undiluted and 1:100 diluted bacterial inocula,were included in each experiment to monitor bacterial growth. When theGI value of the 1:100 inoculum vial reached 30 or greater, ΔGI, thedifference in GI values between the latest GI and the previous readingfor all testing vials was derived. The MIC of a given drug was definedas the lowest concentration at which the ΔGI of the drug vial was lessthan the ΔGI of the 1:100 control. All the experiments were completedwithin 5-8 days.

Data Analysis: The effects of drugs in combination were evaluated basedon GI values using the technique that was previously described.⁹ Synergywas defined as x/y<1/z, where x is the GI of the test vial with thecombination of drugs, y is the lowest GI of the single drugs of thecombination and z is the number of drugs combined. For a two-drugcombination, a quotient of <0.5 was indicative of a synergistic effect,a quotient of 1 indicated no interaction, a quotient of >2 showed anantagonistic effect, and a quotient of <0.75 but >0.5 indicated anadditive effect.

Results

Antimicrobial activity of SQ109 in vitro: We previously determined thatSQ109 MIC is 0.2 μM (0.11 mg/L) by micro-broth dilution or 0.63CM (0.35mg/L) by BACTEC for the laboratory strain H37Rv M. tuberculosis. ³Recently completed determination of SQ109 MIC on more than 30 M.tuberculosis clinical isolates (drug susceptible and drug resistant,including EMB R strains) found the susceptibility of these clinicalstrains to be indistinguishable from H37Rv (MIC range 0.16-0.64 mg/L).These in vitro activities of SQ109 suggest that it is equally activeagainst drug-sensitive and drug-resistant M. tuberculosis, includingstrains resistant to the parent pharmacophore ethambutol.

Interaction of SQ109 with Isoniazid, Streptomycin, Ethambutol, andPyrazinamide:

Using a checkerboard titration, in which series of dilutions of twoantibiotics are studied for effects on bacterial growth inhibition atall possible concentrations, both alone and in combination, the natureof the interaction between the two antibiotics can be determinedalgebraically. The interaction between two antibiotics in combinationcan be described as synergistic, additive, no effect, or antagonistic.In order to translate the experimental data into the types of drug-druginteractions, the quotient x/y (where x is the data obtained when twoantibiotics are in combination and y is the data with the lower value ofthe two agents when tested separately at the same concentration). If thex/y value equals 1, it is interpreted as one of the drugs in combinationis inactive. If x/y is less than 0.5 for a two drug combination, itimplies that the two drugs when used together are more effective thanwhen they are used separately, suggesting synergistic effects. If x/yvalues fail between 0.5 to 0.75, an additive effect may exist betweenthe two drugs, suggesting a weak enhancement between them. If x/y valuesare greater than 2, it suggests antagonistic interactions between thetwo drugs. The window between x/y greater than 1 and less than 2 is thetransition area from no effect to antagonistic. By applying simplealgebra as published and validated elsewhere, we can evaluate thepotential outcome of two drugs in combination in an in vitro assaysystem.

To determine the optimal drug combination(s) that might include SQ109 infuture human efficacy trials, we analyzed the interaction of SQ109 withantibiotics that are currently used to treat TB patients. The MIC ofeach drug is the lowest concentration that inhibits the growth of 99% ofthe bacterial inoculum, thus it is clear that synergistic, additive, orantagonistic effects of combination drugs must be evaluated atindividual drug concentrations that are below the level of the MIC foreffects to be observed. Table 40 (FIG. 67) lists the MIC of eachindividual drug, as well as quotient values (see definition in DataAnalysis, Materials and Methods) for combinations of SQ109 withisoniazid, streptomycin, ethambutol, and pyrazinamide at drugconcentrations below their MIC values. The concentration of each of thedrugs used in combination was expressed as the fraction of the MICvalue. This table includes only the combinations at which synergy oradditive effects were observed. For the drug combinations with noobserved effect, only the quotients obtained from the combination withthe highest sub-optimal dose tested in the experiments were listed.SQ109 showed synergy with isoniazid when both drugs were used at 0.5 MICand showed an additive effect when combined with streptomycin. SQ109 didnot show any positive interaction (additive or synergistic) with eitherethambutol or pyrazinamide, even though the combination with ethambutolwas at the borderline for additivity. No antagonism was observed withany two-drug combination tested in this study.

Interaction of SQ109 with Rifampicin.

When SQ109 was used in combination with rifampicin, we observed markedsynergy between the two drugs, as indicated by the average quotientvalues (Table 41, FIG. 67). This synergy worked both ways: SQ109synergistically enhanced rifampicin activity, and rifampicinsynergistically enhanced SQ109 activity. SQ109 at 0.5 MIC showed synergywith rifampicin at concentrations as low as 0.1 MIC. Synergy was alsoobserved when 0.2 MIC SQ109 was combined with 0.5 MIC rifampicin.Interestingly, the combination with both drug concentrations below 0.5MIC (0.2 MIC SQ109+0.25 MIC rifampicin) showed an additive interaction.

SQ109 and rifampicin interaction in RIF^(R) M. tuberculosis To examinewhether the synergistic interaction between SQ109 and rifampicin alsofacilitated inhibition of drug resistant M. tuberculosis strains, weevaluated drug interactions with RIF^(R) M. tuberculosis clinicalisolates. The rifampicin MIC on RIF^(R) M. tuberculosis isolates 3185and 2482 was 24 mg/L and >100 mg/L, respectively, much higher than theaverage MIC of drug-susceptible M. tuberculosis H37Rv (0.17 mg/L, Table41 legend). The RIF^(R) phenotype did not affect the MIC of SQ109 onthese strains: MIC on both was 0.32 mg/L, the same value as thatdetermined on RIF^(s) M. tuberculosis H37Rv. FIG. 68 shows the GIprofiles of strain 3185 when its susceptibility to rifampicin was testedin the presence of 0.5 MIC of SQ109 (a) or 0.5 MIC ethambutol (b). TheRIF^(R) bacilli grew well in the presence of 0.6 MIC rifampicin (16mg/L, over 90-fold higher than the average MIC determined for M.tuberculosis H37Rv). Addition of 0.5 MIC SQ109 (0.16 mg/L) torifampicin-treated RIF^(R) bacteria inhibited greater than 99% growth.Rifampicin dose-dependent inhibition of RIF^(R) M. tuberculosis growthin the presence of SQ109 decreased with decreasing rifampicinconcentration in the test vials. The x/y quotients for rifampicin at 16,12, and 8 mg/L were 0.35, 0.40, and 0.48, respectively, indicatingsynergistic interaction between SQ109 and rifampicin. An additiveinteraction was observed at 6 and 4 mg/L rifampicin (x/y−0.55 and 0.64,respectively). This synergy was not observed when 0.5 MIC ethambutol, adifferent diamine antibiotic, was used in combination with rifampicin(FIG. 68 (b)).

Experiments with RIF^(R) strain 3185 were repeated with a differentRIF^(R) M. tuberculosis, strain 2482, whose rifampicin resistance waseven more profound: MIC>100 mg/L. As shown in FIG. 68, the bacteria grewmodestly in the presence of 100 mg/L rifampicin. Adding 0.5 MIC SQ109 tothis culture inhibited growth more than 99%. The x/y quotients forrifampicin at 100, 50, and 25 mg/L were 0.33, 0.39, and 0.497,respectively, indicating synergistic interaction between SQ109 andrifampicin. Additive interactions were observed at 12.5 and 6.25 mg/Lrifampicin (x/y=0.62 and 0.71, respectively). In addition, an additiveeffect was also observed at 0.25 MIC SQ109 (0.08 mg/L) and 10 mg/Lrifampicin (x/y=0.66). Again, 0.5 MIC ethambutol did not show anyadditive or synergistic activity with rifampicin on strain 2482 (datanot shown). In both cases, the concentration to MIC ratio at which thesynergy between SQ109 and rifampicin was observed was similar to thoseobtained for the RIF^(s) strain, implying that the synergisticinteraction was independent of drug susceptibility status. These resultsstrongly suggest that the synergy between SQ109 and rifampicin wasspecific for this combination of drugs, and that the enhanced druginteractions inhibited M. tuberculosis functions in both RIF^(s) andRIF^(R) strains.

Discussion

Multi-drug therapy is essential to cure TB infections and avoid theemergence of drug-resistant bacteria. Any new anti-TB drug candidateneeds to be evaluated for combination drug interactions to optimizeindividual drug activity and avoid drug antagonism. In this study wereport our findings on the interaction of SQ109, a new diamineantitubercular drug candidate, with commonly used anti-TB drugs invitro. The possible interactions that we could measure in these studiesusing growth inhibition included antagonistic, synergistic, additive orno effect at all. We found no antagonistic interactions between SQ109and any of the five front-line TB drugs tested in this study. Thecombinations of [SQ109+ethambutol] or [SQ109+pyrazinamide] showed nointeractions, positive or negative, and the effects on growth of M.tuberculosis of these combinations were indistinguishable from thoseobtained with single drug treatment. An additive interaction wasobserved between SQ109 and streptomycin at certain concentrations, butno synergy was observed. In contrast, SQ109 showed synergy with twodifferent front-line drugs: isoniazid and rifampicin. The synergisticinteractions between SQ109 and rifampicin were particularly interesting,and quite potent: greater than 99% inhibition of M. tuberculosis growthwas achieved at very low concentrations of the individual drugs, 0.1 MICrifampicin+0.5 MIC SQ109. The in vitro synergy results described in thispaper were consistent with in vivo studies (Nikonenko, et al., inpreparation) that demonstrated drug synergy in experimental animalsinfected with M. tuberculosis when SQ109 was combined with rifampicinand isoniazid. These in vivo results showed enhanced bactericidalactivity and faster elimination of M. tuberculosis by SQ109-containingdrug combinations compared to similar combinations containingethambutol. Together, the in vitro and in vivo data presented in thesetwo studies can guide us in achieving the most efficacious drugcombination design in future SQ109 clinical trials for treatment ofactive TB disease.

Interestingly, data in the present study also showed that SQ109 is fullyactive against RIF^(R) M. tuberculosis clinical isolates. This isconsistent with the recent results obtained from the study of over 30drug-sensitive and drug-resistant M. tuberculosis, where the strains hadthe same SQ109 MIC (range 0.16-0.64 mg/L), suggesting that there is nopre-existing resistance to this compound. Synergy of SQ109 withrifampicin was also observed when tested against RIF^(R) isolates. At0.5 MIC, SQ109 was able to increase rifampicin activity against theresistant organisms in a dose-dependent manner. This activity was notobserved with 0.5 MIC ethambutol, suggesting that a specific interactionbetween SQ109 and rifampicin is likely responsible for the observation.Although this observation may not have direct clinical relevance, itpoints out additional differences between SQ109 and its parentpharmacophore, ethambutol, and could be an interesting experimentalmodel to tease out SQ109 actions on M. tuberculosis.

A definitive explanation for the profound synergy between rifampicin andSQ109 against M. tuberculosis is not yet available. Hypothetically,though not wishing to be bound by the following theory, the synergisticinteraction could result from the differences in drug action. Althoughthe precise target of SQ109 is unknown, it does affect mycobacterialcell wall synthesis. Perhaps the effect of SQ109 on the mycobacterialcell wall results in increasing permeability, allowing more rifampicinto enter the bacteria. Subinhibitory concentrations of cell wallinhibitors such as ethambutol were shown to increase bactericidalactivity of clarithromycin for M. tuberculosis, presumably by decreasingthe permeability barrier for drug entry. However, ethambutol, acell-wall inhibitor, did not show any synergy when used in combinationwith rifampicin in our experiments with RIFS H37Rv or the RIF R M.tuberculosis clinical isolates (FIG. 68) or in other drug susceptiblestrains suggesting that just any effect on the cell wall structure ofmycobacteria does not necessarily contribute to the synergisticinteraction between rifampicin and other drugs. In addition, rifampicinis already known to rapidly penetrate the hydrophobic cell wall ofmycobacteria due to its lipophilic nature. As a result, the effect(s) oncell wall permeability by SQ109 is not likely to be the sole contributorto the observed synergy between rifampicin and SQ109 in vitro.

It is also possible that the expression level of the currently unknownprimary target of SQ109 is tightly regulated at the transcriptionallevel. Rifampicin is an inhibitor of DNA transcription. Sincetranscripts of mRNA of various genes differ in half-life, inhibition oftranscription could have profound effects on those transcripts withshorter half-life than those with longer half life. Thus, rifampicintreatment, even at suboptimal concentrations, could exert a noticeableeffect on the level of a short-lived mRNA target for SQ109 activity.However, this hypothesis is inconsistent with the observation thatsynergy between the two drugs was not affected by the RIF^(R) phenotypeof the M. tuberculosis strains. This suggests that other effects ofrifampicin, rather than only its primary action as an antibiotic,contributed to its synergistic interaction with SQ109. That rifampicinantibiotic activity might certainly be a contributing factor, butperhaps not the only factor that determined the interesting interactionwith SQ109, was suggested by data on the MIC of RIF^(R) strains. As theMIC of rifampicin becomes greater in RIF^(R) as compared with RIF^(s)strains, the concentrations of rifampicin showing synergy with the sameamount of SQ109 are also greater, even though the combinations expressedin terms of fractions of MIC were similar between the strains studied.To fully evaluate the effect(s) of rifampicin transcription inhibitionon SQ109 activity requires the identification of the SQ109 target(s),which is ongoing work in our laboratory.

Another possible effect of rifampicin on the drug synergy interactionbetween rifampicin and SQ109 is the rifampicin action as an efficientinducer of cytochrome P450 (CYP). The CYP are a superfamily ofheme-containing enzymes involved in a wide array of NADPH/NADH-dependentreactions, and they play pivotal roles in biosynthesis of compounds suchas sterols, steroids, and fatty acids, as well as in detoxification ofxenobiotics and chemicals. Rifampicin is a potent inducer of a varietyof CYP in human hepatocytes, as well as in peripheral blood lymphocytes.

The complete genome of M. tuberculosis reveals at least 20 CYP, butprecise functions for these genes remain to be elucidated. Like theirmammalian counterparts, the mycobacterial CYP were induced byrifampicin. Ramachandran and Gurumurthy determined CYP activity presentin bacterial membrane fractions extracted from rifampicin-treated M.smegmatis and M. tuberculosis. In both cases, the CYP activities in thetreated fractions were elevated as compared to the untreated controls,and the increase in CYP activity was statistically significant. Inparallel, they found that isoniazid did not induce CYP activity in itstreated fractions. The ability of rifampicin to induce CYP in M.tuberculosis may contribute to its synergy with SQ109. In this case,instead of inactivating the active compounds, CYP may in fact activateSQ109 by producing oxidized metabolites of SQ109 within M. tuberculosis.

Recently obtained data on SQ109 metabolism showed that SQ109 wasmetabolized rapidly after incubation with human liver microsomes: only8% SQ109 remained after 40 min incubation. Analysis of SQ109 metabolitesproduced by the action of the microsomes revealed 4 chemical groupsbased on molecular mass. Two of the groups contained oxidizedmetabolites. Furthermore, the study found that SQ109 was metabolized byhuman CYP2D6 and CYP2C19 to generate these metabolites. Based on thefinding that SQ109 was metabolized rapidly by CYP, it is possible thatits antimycobacterial activity may come from one of its metabolites. Itis conceivable that SQ109 is a prodrug and requires activation bymycobacterial CYP. We speculate that when SQ109 is used in combinationwith rifampicin, the latter induces certain CYP activity within theMycobacteria. Elevated CYP activity activates the SQ109 prodrug moreefficiently, resulting in an apparent synergistic activity of the twodrugs. In fact, the enhanced activity seen with the combination ofSQ109+rifampicin may be the more efficient and effective activity of anSQ109 active metabolite, rather than enhanced activity of rifampicinitself.

SQ109 has a very narrow spectrum: it is active against M. tuberculosisand M. bovis BCG, but much less active against M. smegmatis and M. avium(unpublished data). By comparing the putative CYP open reading framespresent in various mycobacterial genomes, Kelly et al showed there wereseveral CYP that are unique in M. tuberculosis and M. bovis. These CYPare strong candidates for actors that might be responsible forconverting SQ109 to an active metabolite(s) within M. tuberculosis, andthey are the subjects of ongoing investigations.

Example XXIX Multidrug Study

The present experiment will investigate the efficacy of Isoniazid(INH)+Rifampin (RIF)+Pyrazinamide (PZA)+SQ109 (INH/RIF/PZA/SQ109) forelimination of M. tuberculosis in lung during intensive phase therapy inanimal modeling experiments (see FIG. 70) compared to standard DOTSINH+RIF+PZA+Ethambutol (EMB) (INH/RIF/PZA/EMB)

The preferred Phase II Study will be a prospective, multi-center,double-blind, randomized, placebo-controlled, clinical study of SQ109designed to:

-   -   Determine the safety and tolerability of SQ109 in male and        female volunteer tuberculosis patients with a positive sputum        culture during two (2) month oral administration for up to 0-50        mg/day (TBD) SQ109 in combination with INH/RIF/PZA. (Primary        Objective)    -   Evaluate the minimal dose of SQ109 in tuberculosis patients        which will convert patients to sputum culture negative by or        within 2 months (Primary Objective).

An animal (mouse) experiment will be conducted to increase SQ109 dosewhile holding INH/RIF/PZA at the standard DOTS dose to mimic the designof the present study and predict potential human doses.

Preliminary INH, RIF & PZA drug interaction studies, including ADME inmice and in vitro drug interactions (microsomes), are also beingconducted.

Alternate Multidose (5 Day) EBA Study

An alternative Phase II multidose study would be an ‘Early BactericidalAssay (EBA)’ study as proposed by Dennis Mitchison for new TB drugdevelopment and as conducted by Stephen Gillespie (London) and. In thesestudies, antibiotic monotherapy is conducted for a short period of timewhile monitoring numbers of bacteria in sputum. These studies arecapable of determining a minimum effective dose of a single antibioticto reduce bacteria in sputum and can expand the safety profile of SQ109;however these studies are not capable of providing efficacy data forcombination therapy. It is anticipated that tuberculosis will, for theforeseeable future, be treated with at least 3 drugs with differentmodes of action.

The alternative multidose (5 day) EBA study would be a randomized, open,clinical study of SQ109 designed to determine the clinical efficacy ofSQ109 in patients with pulmonary tuberculosis based on EBA of SQ109 andto further evaluate its safety. EBA studies provide a fast and economicway to evaluate the clinical efficacy of potential agents for thetreatment of tuberculosis in a brief period (3-14 days, usually 5 days)of study medication monotherapy.

Approximately one hundred patients (see criteria below) will be orallytreated with either SQ109 once daily (n=40) or 6-mg/kg INH (n=10) for 5days. The study treatment period will be followed by a standardtherapeutic regimen for 6 months. The dose of SQ109 for this study willbe determined on based on other studies, such as the highest amount ofthe drug that can be taken with good PK parameters and without harmfulside effects, also taking into a consideration the therapeutic doseobtained in the efficacy studies with laboratory animals (30 mg/m²).

Enrollment Inclusion Criteria:

Adults, male or female, aged 18-60

Newly diagnosed initial episodes of pulmonary tuberculosis.

Chest X-ray and clinical findings consistent with tuberculosis.

Sputum smear-positive patients will be eligible for enrollment. Thediagnosis of tuberculosis must be also confirmed by culture. AFB smearpositive patients found later not to have TB (i.e. those withnon-tuberculous mycobacterial disease) and those without cultureconfirmation will be removed from the study.

Enrollment Exclusion Criteria:

Pregnant or breastfeeding

HIV-infected

History of prior tuberculosis or history of previous tuberculosistreatment

Cavitary tuberculosis on initial chest X-ray (taken within 14 days ofstudy entry)

Exposure to person(s) with known drug resistant tuberculosis

Patients with drug resistant tuberculosis (resistance to INH, RIF, PZAor EMB)

Patients receiving chronic steroids or other immunosuppressivemedications

Extra-pulmonary tuberculosis

Sputum and blood will be collected 2 days before treatment (baseline)and after 2 and 5 days of study treatment. Hematological andbiochemistry parameters will be assessed simultaneously. The count ofCFU per milliliter will be determined as described (Jindani A, Doré CJ,Mitchison DA, American Journal of Respiratory and Critical Care Medicine2003 Vol 167. pp. 1348-1354). The EBA will be calculated for eachpatient by the formula (log CFU/ml_(day0)−log CFU/ml_(day5))/5 and willbe reported as the mean±standard deviation (SD). As recommended byseveral authors, we will define the EBA as the decrease in log₁₀ CFU permilliliter of sputum per day during the first 5 days of treatment.

Example XXX Interaction of SQ109 with INH, SM, EMB, and PZA In Vitro

To determine the optimal drug combination that includes SQ109 for futureefficacy trials in humans, we analyzed the interaction of SQ109 withantibiotics that are currently used to treat TB patients. The MIC ofeach drug is the lowest concentration that kills or inhibits the growthof 99 percent of the bacterial inoculum. It is clear that synergistic,additive, or antagonistic effects of combination drugs must be evaluatedat individual drug concentrations that are below the level of the MICfor effects to be observed. Table 41 lists the MIC of each individualdrug, as well as quotient values for combinations of SQ109 with INH,STR, EMB, and PZA at drug concentrations below their MIC values. Theconcentration of each of the drugs used in combination was expressed asthe fraction of the MIC value. In general, synergy effects are observedwhen quotient values of a combination doesn't exceed 0.5, additiveeffects when the quotient values are within a range of 0.5-0.75.

Drugs INH SM MOX RIF Folds of MIC ½ ½ ⅕ ½ 1/20 1/10 ¼ ½ SQ109 1/20 1 1 11 1 1 1 0.32 (folds 1/10 1 0.98 1 1 1 1 1 0.16 of ⅕ 0.83 0.74 1 0.96 1 11 0.4 MIC) ½ 0.45 0.57 0.51 0.45 0.61 0.29 0.23 0.12Table 41. Synergy quotients for SQ109 tested in two-drug combinationswith INH, SM, EMB, or PZA MIC: INH: 0.05 μg/ml, SM: 0.25 μg/ml, EMB:1.25 μg/ml, PZA: 100 μg/ml, SQ109: 0.32 μg/ml or 0.64 μg/ml.

The results of this synergy experiment care be summarized as follows:

-   -   SQ109 showed synergy with INH in vitro when both drugs used at ½        MIC.    -   SQ109 has no interaction with EMB and only additive interaction        with SM.    -   SQ109 showed marked synergy with ½ MIC RIF at all concentrations        and with lesser concentrations of RIF at ½ MIC SQ109.    -   SQ109 has no antagonistic interaction with any of the four drugs        tested at alldose combinations.

When SQ109 was used in combination with RIF, we observed marked synergybetween the two drugs, as indicated by the average quotient values(Table 41, and FIG. 71).

Example XXXI SQ109 and RIF Interaction in RIF^(R) M. tuberculosis

To examine whether the synergistic interaction between SQ109 and RIFalso facilitated killing of drug resistant M. tuberculosis strains, weevaluated drug interactions with RIF^(R) M. tuberculosis clinicalisolates. The RIF MIC on RIF^(R) M. tuberculosis isolates 3185 and 2482was 24 μg/ml and >100 μg/ml, respectively, much higher than the averageMIC of drug-susceptible M. tuberculosis H37Rv (0.17 μg/ml). The RIF^(R)phenotype did not affect the MIC of SQ109 on these strains: MIC on bothwas 0.32 μg/ml, the same value as that determined on RIF^(s) M.tuberculosis H37Rv. We have found (FIG. 72) that for both strains, theconcentration to MIC ratio at which the synergy between SQ109 and RIFwas observed was similar to those obtained for the RIF^(s) strain,implying that the synergistic interaction was independent of drugsusceptibility status. These results strongly suggest that the synergybetween SQ109 and RIF was specific for this combination of drugs, andthat the enhanced interaction to kill M. tuberculosis functions in bothRIF^(s) and RIF^(R) strains. No synergy was observed when 0.5 MIC EMBwas used in combination with RIF.

FIG. 72 provides the growth profile of RIF^(R) M. tuberculosis isolate2482 treated with RIF and SQ109. The experiment was carried out inBACTEC 460. The MIC of RIF and SQ109 in Strain 2482 were >100 μg/ml and0.32 μg/ml, respectively.

Combination Therapy; Studies in Mice.

We have carried out in vivo studies where SQ109 was tested for itsefficacy in combination with other anti-TB drugs: Rifampin, Isoniazid,Ethambutol, Pyrazinamide, Moxifloxacin. At first, effectiveness of thedrug combinations was studied in a rapid mouse model (developed bySequella' scientist Dr. Boris Nikonenko) that allows to predict quicklyand with sufficient accuracy the drug's efficacy based on its ability toprevent body weight loss in the infected animals, one of the signs of TBseverity.

Briefly, mice were inoculated iv with 10⁶ CFU of virulent M.tuberculosis H37Rv to develop a rapid and progressive TB disease.Chemotherapy was initiated 7d after inoculation and continued for 10days. Mice treated with a single drug (SQ109, Rif, INH, EMB, PZA, andMoxi), as well as uninfected animals and infected untreated placebo,were used as the controls. In this model, all standard drugs were usedat doses below their most efficacious in order to see the effect (whenused at their therapeutic doses, drug-treated mice did not looseweight): Rif was studied at 2 mg/kg, INH at 1 mg/kg, EMB at 10 mg/kg,Moxi at 10 mg/kg, PZA at 50 mg/kg. Body weights of mice in all groupswere monitored starting from time 0. By 10d, infected placebo controlmice started to lose weight; by 20d mice in this group lost more than25% of their body weight.

The results of the Day 21 (FIG. 73) demonstrate enhanced activity ofSQ109 in combination with Rifampin and INH. SQ109-Rif combination fullyprevented body weight loss during period of chemotherapy. Moreover,SQ109-Rif combination significantly prolongs therapeutic effect evenafter the chemotherapy withdraw, FIG. 73. No effect was seen inSQ109-Ethambutol combination, slight improvement was obtained forSQ109-PZA that may be attributed to SQ109 efficacy alone. Anantagonistic effect was demonstrated for SQ109-Moxi combination that isin contrary to the results obtained in vitro. No improvement has beenseen in Rif-EMB combination. FIG. 73 provides the results of a rapidmodel, combination therapy study, day 21. C3H female mice were infectedi.v. with 10⁶ CFU M. tuberculosis H37Rv (Pasteur) previously passedthrough mice. 7 days following inoculation chemotherapy with anti-TBdrugs were initiated and continued till day 21.

Example XXXII In Vivo Potency of Sequella's Drug Candidates SQ109,SQ609, and SQ73 Tested as a Combination

As part of our efforts to develop a new regimen for treatment oftuberculosis, we studied a combination of SQ109 (at 10 mg/kg) withSequella's potential drug candidates, -dipiperidine SQ609 (at 10 mg/kg)and 1,2-ethylenediamine SQ73 (at 5 mg/kg), FIG. 74, in a mouse model ofchronic TB infection, FIG. 75.

The compounds were used in the following doses: SQ109 at 10 mg/kg; SQ609at 10 mg/kg, SQ73 at 5 mg/kg, totaling overall dose of 25 mg/kg.Activity of this drug combination was compared to the efficacy of one ofthe most efficacious anti-TB drugs isoniazid (INH) which was used ascontrol in this study at 25 mg/kg.

Mice were infected with low dose of M. tuberculosis H37Rv andchemotherapy was initiated 4 weeks following infection and continued for2 weeks.

In this study, SQ609-SQ109-SQ73 combination demonstrated similaractivity to INH at it's the most efficacious dose.

FIG. 75 provides the results of a chronic TB study. C57BL/6 female micewere inoculated i.v. with 10⁴ CFU M. tuberculosis H37Rv. Chemotherapywas initiated four weeks following the infection and continued for 2weeks. One group of mice (6 mice per group) was tested for each controldrug and the drug combination. After 2 weeks of treatment mice weresacrificed; lungs homogenates in sterile 2 ml PBS with 0.05% Tween-80were plated in 10-fold serial dilutions on 7H10 agar dishes, and wereincubated at 37° C. CFU were calculated after 3 wk of growth. INH wasused at 25 mg/kg, SQ109 at 10 and 25 mg/kg, SQ609 at 10 mg/kg;combination (“Sum” on the chart): SQ109 at 10 mg/kg; SQ609 at 10 mg/kg,SQ73 at 5 mg/kg. Statistic analysis was done using the ANOVA test:significance of any differences was estimated by Student's T-test andp<0.05 was considered statistically significant.

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1. A composition comprising a substituted ethylene diamine compound ofthe formula

wherein R₄ is selected from H, alkyl, aryl, heteroatom substituted alkyland aryl alkenyl, alkynyl, aralkyl, aralkynyl, cycloalkyl, cycloalkenyl;and wherein R₁, R₂ and R₃ are independently selected from H, alkyl,aryl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl, cycloalkyl,cycloalkenyl, heteroalkyl, heteroaryl, halide, alkoxy, aryloxy,alkylthio, arylthio, silyl, siloxy, amino; or wherein R₁ is selectedfrom H, alkyl, aryl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl,cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl, halide, alkoxy,aryloxy, alkylthio, arylthio, silyl, siloxy, amino, and NR₂R₂ is derivedfrom a cyclic secondary amine, further comprising an antimicrobial,antibacterial, antimycological, antiparasitic, antiviral agent.
 2. Thecomposition of claim 1, wherein the antibacterial agent comprises anantitubercular agent.
 3. The composition of claim 3, wherein theantitubercular agent comprises rifampicin, isoniazid, pyrazinamide,moxifloxacin and ethambutol.
 4. The composition of claim 1, whereinNHR₁, or NR₂R₃ of the substituted ethylene diamine has the chemicalstructure


5. The composition of claim 4, wherein the substituted ethylene diaminecompound is selected from


6. The composition of claim 1, wherein NHR₁ or NR₂R₃ of the substitutedethylene diamine has the chemical structure


7. The composition of claim 6, wherein the substituted ethylene diaminecompound is selected from


8. The composition of claim 1, wherein NHR₁ or NR₂R₃ of the substitutedethylene diamine has the chemical structure


9. The composition of claim 8, wherein the substituted ethylene diaminecompound is selected from


10. The composition of claim 1, wherein NHR₁ or NR₂R₃ of the substitutedethylene diamine has the chemical structure


11. The composition of claim 10, wherein the substituted ethylenediamine compound is selected from


12. The composition of claim 11, wherein the substituted ethylenediamine compound is


13. The composition of claim 1, wherein the substituted ethylene diaminecompound is selected from


14. A method of preparing a substituted ethylene diamine compound of theformula

wherein R₄ is selected from H, alkyl, aryl, alkenyl, alkynyl, aralkyl,aralkynyl, cycloalkyl, cycloalkenyl; and wherein R₁, R₂ and R₃ areindependently selected from H, alkyl, aryl, alkenyl, alkynyl, aralkyl,aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl,halide, alkoxy, aryloxy, alkylthio, arylthio, silyl, siloxy, amino; orwherein R₁ is selected from H, alkyl, aryl, alkenyl, alkynyl, aralkyl,aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl,halide, alkoxy, aryloxy, alkylthio, arylthio, silyl, siloxy, amino, andNR₂R₂ is derived from a cyclic secondary amine; comprising activating asolid-support resin containing hydroxyl groups with a halo-donatingreagent in the presence of base to produce a solid-support resincontaining halo groups; displacing the halo groups with an initialprimary amine to produce a solid-support resin containing amine groups;acylating the amine groups with a halo-acylhalide in the presence of abasic compound, or with a halo-acylacid in the presence of base, toproduce a solid-support resin containing α-haloacetyl amide groups;displacing α-halo groups of the α-haloacetyl amides with a secondary orsubsequent primary amine to produce a solid-support resin containingα-amine imide groups; reducing the carbonyl moiety on the α-amine imidegroups with a reducing agent to produce a solid-support resin containingtwo amine groups separated by two carbon atoms; cleaving the aminegroups separated by two carbon atoms from the solid support resin in thepresence of acid to produce the substituted ethylene diamine compound.15. The method of claim 12, wherein the initial primary amine is R₁NH₂.16. The method of claim 12, wherein the secondary or subsequent primaryamine is R₂R₃HN.
 17. A method of treating disease caused by aninfectious agent comprising administering an effective amount of acomposition comprising substituted ethylene diamine compound of theformula:

wherein R₄ is selected from H, alkyl, aryl, alkenyl, alkynyl, aralkyl,aralkynyl, cycloalkyl, cycloalkenyl; and wherein R₁, R₂ and R₃ areindependently selected from H, alkyl, aryl, alkenyl, alkynyl, aralkyl,aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl,halide, alkoxy, aryloxy, alkylthio, arylthio, silyl, siloxy, amino; orwherein R₁ is selected from H, alkyl, aryl, alkenyl, alkynyl, aralkyl,aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl,halide, alkoxy, aryloxy, alkylthio, arylthio, silyl, siloxy, amino, andNR₂R₂ is derived from a cyclic secondary amine further comprising anantimicrobial, antibacterial, antimycological, antiparasitic, antiviralagent.
 18. The composition of claim 17, wherein the antibacterial agentcomprises an antitubercular agent.
 19. The composition of claim 17,wherein the antitubercular agent comprises rifampicin, isoniazid,pyrazinamide, moxifloxacin and ethambutol.
 20. The method of claim 17,wherein the infectious agent comprises a bacterial, mycological,parasitic, or viral agent.
 21. The method of claim 20, wherein thebacterial agent comprises M. tuberculosis, M. avium-intracellulare, M.kansarii, M. fortuitum, M. chelonae, M. leprae, M. africanum, M.microti, M. avium paratuberculosis, M. intracellulare, M. scrofulaceum,M. xenopi, M. marinum, or M. ulcerans.
 22. The method claim 17, whereinthe disease comprises tuberculosis or Crohn's disease.
 23. The methodclaim 17, wherein the substituted ethylene diamine compound is


24. The method of claim 23, further comprising a pharmaceutical carrier.25. A composition comprising, a substituted ethylene diamine compoundcomprising,


26. A method of preparing a substituted ethylene diamine compound of theformula

wherein R₄ is selected from H, alkyl, aryl, heteroatom substituted alkyland aryl, alkenyl, alkynyl, aralkyl, aralkynyl, cycloalkyl,cycloalkenyl; and wherein it, R₂ and R₃ are independently selected fromH, alkyl, aryl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl,cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl, halide, alkoxy,aryloxy, alkylthio, arylthio, silyl, siloxy, amino; comprisingactivating a solid-support resin containing hydroxyl groups with ahalo-donating reagent in the presence of base to produce a solid-supportresin containing halo groups; displacing the halo groups with an initialprimary amine to produce a solid-support resin containing amine groups;acylating the amine groups with a FMOC protected amino acid in thepresence of a coupling reagent and a base, followed by removal of FMOCprotecting group to produce a solid-support resin containing α-aminoacetamide groups; modification of α-amino groups of the α-aminoacetamide groups with a carbonyl compound to produce a solid-supportresin containing corresponding derivative of α-amino acetamide groups;reducing the carbonyl moiety on the amide groups with a reducing agentto produce a solid-support resin containing two amine groups separatedby two carbon atoms; and cleaving the amine groups separated by twocarbon atoms from the solid support resin in the presence of acid toproduce the substituted ethylene diamine compound.
 27. A method fortreating an infectious disease comprising administering apharmaceutically effective amount of the composition in claim
 25. 28. Acomposition comprising a symmetrical substituted ethylene diaminecompound of the formula

wherein R₄ is selected from H, alkyl, aryl, heteroatom substituted alkyland aryl alkenyl, alkynyl, aralkyl, aralkynyl, cycloalkyl, cycloalkenyl;and wherein R₁, R₂ and R₃ are independently selected from H, alkyl,aryl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl, cycloalkyl,cycloalkenyl, heteroalkyl, heteroaryl, halide, alkoxy, aryloxy,alkylthio, arylthio, silyl, siloxy, amino; or wherein R₁ is selectedfrom H, alkyl, aryl, alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl,cycloalkyl, cycloalkenyl, heteroalkyl, heteroaryl, halide, alkoxy,aryloxy, alkylthio, arylthio, silyl, siloxy, amino, and NR₂R₂ is derivedfrom a cyclic secondary amine, further comprising an antimicrobial,antibacterial, antimycological, antiparasitic, or antiviral agent. 29.The composition of claim 28, wherein the antibacterial agent comprisesan antitubercular agent.
 30. The composition of claim 29, wherein theantitubercular agent comprises rifampicin, isoniazid, pyrazinamide,moxifloxacin and ethambutol.